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Abstract:

A method for modulating heat loss in a neonatal mammalian subject is
disclosed herein that includes providing exogenous brown adipocytes, or
precursors thereof, to an internal site in the neonatal mammalian
subject.

Claims:

1. A method for modulating heat loss in a neonatal mammalian subject
comprising: providing exogenous brown adipocytes, or precursors thereof,
to an internal site in the neonatal mammalian subject.

8. The method of claim 1, wherein modulating heat loss includes reducing
heat loss in the neonatal mammalian subject by providing the exogenous
brown adipocytes, or the precursors thereof, to the internal site in the
neonatal mammalian subject.

11. The method of claim 1, wherein the internal site includes one or more
of a subcutaneous site, scapular site, axillary site, thoracic site,
abdominal site, or blood vessel site of the neonatal mammalian subject.

12. The method of claim 1, wherein the brown adipocytes or the precursors
thereof derive from one or more of cell donors, tissue donors, tissue
culture stock, cell lines, or genetically manipulated cells.

13. The method of claim 12, wherein the genetically manipulated cells
include an exogenous DNA sequence encoding one or more of a mammalian UCP
polypeptide or a PRDM16 polypeptide.

14. The method of claim 12, wherein the one or more cell donors or tissue
donors include a genetically related donor of the neonatal mammalian
subject.

15. The method of claim 14, wherein the genetically related donor is a
mother, father, sibling, grandparent, aunt, or uncle of the neonatal
mammalian subject.

16. The method of claim 1, wherein the exogenous brown adipocytes or the
precursors thereof derive from one or more of an autologous tissue,
allogeneic tissue, or xenogeneic tissue.

17. The method of claim 1, wherein the exogenous brown adipocytes or the
precursors thereof derive from a neonatal-associated tissue.

18. The method of claim 1, wherein the exogenous brown adipocytes or the
precursors thereof derive from one or more of adipocytes, pre-adipocytes,
stem cells, cord blood cells, placental cells, myoblasts, or bone marrow
cells.

19. The method of claim 1, comprising expanding, maturing, or
differentiating the exogenous brown adipocytes or the precursors thereof
in vitro.

20. The method of claim 19, comprising providing one of more of
differentiation factors or growth factors in vitro to the exogenous brown
adipocytes or the precursors thereof.

21. The method of claim 1, wherein the brown adipocytes, or the
precursors thereof include one or more detectable markers incorporated
with the brown adipocytes or the precursors thereof.

22. The method of claim 1, wherein providing the brown adipocytes or the
precursors thereof to the internal site comprises injecting the brown
adipocytes or the precursors thereof.

23. The method of claim 22, comprising injecting the brown adipocytes or
the precursors thereof in a pharmaceutically acceptable carrier.

24. The method of claim 1, wherein providing the brown adipocytes or the
precursors thereof to the internal site comprises implanting the brown
adipocytes or the precursors thereof.

25. The method of claim 1, comprising providing the brown adipocytes or
the precursors thereof in a pharmaceutically acceptable carrier.

26. The method of claim 1, comprising providing the brown adipocytes or
the precursors thereof in one or more biocompatible carriers.

27. The method of claim 26, comprising encapsulating the brown adipocytes
or the precursors thereof.

28. The method of claim 26, comprising providing the brown adipocytes or
the precursors thereof in an immunoisolating material.

40. The method of claim 33, wherein modulating heat loss includes
reducing heat loss in the neonatal mammalian subject by providing the
exogenous brown adipocytes, or the precursors thereof, to the internal
site in the neonatal mammalian subject.

44. The method of claim 33, wherein the brown adipocytes or the
precursors thereof derive from one or more of cell donors, tissue donors,
tissue culture stock, cell lines, or genetically manipulated cells.

45. The method of claim 44, wherein the genetically manipulated cells
include an exogenous DNA sequence encoding one or more of a mammalian.
UCP polypeptide or a PRDM16 polypeptide.

46. The method of claim 44, wherein the one or more cell donors or tissue
donors include a genetically related donor of the neonatal mammalian
subject.

47. (canceled)

48. The method of claim 33, wherein the exogenous brown adipocytes or the
precursors thereof derive from one or more of an autologous tissue,
allogeneic tissue, or xenogeneic tissue.

49. The method of claim 33, wherein the exogenous brown adipocytes or the
precursors thereof derive from a neonatal-associated tissue.

50. The method of claim 33, wherein the exogenous brown adipocytes or the
precursors thereof derive from one or more of adipocytes, pre-adipocytes,
stem cells, cord blood cells, placental cells, myoblasts, or bone marrow
cells.

51. The method of claim 33, comprising expanding, maturing or
differentiating the exogenous brown adipocytes or the precursors thereof
in vitro.

52. The method of claim 51, comprising providing one or more of
differentiation factors or growth factors in vitro to the exogenous,
brown adipocytes or the precursors thereof.

53. The method of claim 33, wherein the brown adipocytes or the
precursors thereof include one or more detectable markers incorporated
with the brown adipocytes or the precursors thereof.

54. The method of claim 33, wherein providing the brown adipocytes or the
precursors thereof to the internal site comprises injecting the brown
adipocytes or the precursors thereof.

55. The method of claim 33, comprising injecting the brown adipocytes or
the precursors thereof in a pharmaceutically acceptable carrier.

56. The method of claim 33, wherein providing the brown adipocytes or the
precursors thereof to the internal site comprises implanting the brown
adipocytes or the precursors thereof.

57. (canceled)

58. The method of claim 33, comprising providing the brown adipocytes or
the one or more precursors thereof in one or more biocompatible carriers.

59. The method of claim 58, comprising encapsulating the brown adipocytes
or the precursors thereof.

60. The method of claim 58, comprising providing the brown adipocytes or
the one or more precursors thereof in an immunoisolating carrier.

61.-64. (canceled)

Description:

SUMMARY

[0001] Methods are disclosed herein for modulating heat loss in a neonatal
mammalian subject. The method includes providing exogenous brown
adipocytes, or precursors thereof, to an internal site in the neonatal
mammalian subject. In the method disclosed herein, providing brown
adipocytes can include providing pre-adipocytes or providing precursor
cells of exogenous brown adipocytes to an internal site in the neonatal
mammalian subject. The method can further include providing factors that
promote at least one of adipocyte proliferation, adipocyte
differentiation, or adipose angiogenesis. The factors can include, but
are not limited to, differentiation factors, growth factors, or
angiogenic factors. The factors that support or enhance proliferation can
be configured to induce differentiation of the pre-adipocytes or
adipocyte precursors to brown adipocytes. The internal site for placement
of brown adipocytes in the neonatal mammalian subject can include one or
more of a subcutaneous site, scapular site, axillary site, thoracic site,
abdominal site, or blood vessel site of the neonatal mammalian subject.
In some aspects, the brown adipocytes, or precursors thereof, can be
substantially purified. The neonatal mammalian subject can include, but
is not limited to, a human, equine, bovine, ovine, swine, rodent,
lagomorph, canine, or feline. The neonatal mammalian subject can include
a neonatal mammalian subject born preterm.

[0002] In some aspects, modulating heat loss includes reducing heat loss
in the neonatal mammalian subject by providing the exogenous brown
adipocytes, or the precursors thereof, to the internal site in the
neonatal mammalian subject. The precursors can include, but are not
limited to, one or more of stem cells, totipotent stem cells, multipotent
stem cells, pluripotent stem cells, oligopotent stem cells, embryonic
stem cells, de-differentiated stem cells, trans-differentiated stem
cells, mesenchymal stem cells, adipose-derived stem cells, adipocyte
progenitor cells, pre-adipocytes, myoblasts, muscle-derived stem cells,
or bone marrow-derived stem cells. The exogenous brown adipocytes can
include mature brown adipocytes. The internal site can include, but is
not limited to, one or more of a subcutaneous site, scapular site,
axillary site, thoracic site, abdominal site, or blood vessel site of the
neonatal mammalian subject. The brown adipocytes or the precursors
thereof can be derived from one or more of cell donors, tissue donors,
tissue culture stock, cell lines, or genetically manipulated cells. The
genetically manipulated cells can include an exogenous DNA sequence
encoding one or more of a mammalian UCP polypeptide or a PRDM16
polypeptide.

[0003] The one or more cell donors or tissue donors can include a
genetically related donor of the neonatal mammalian subject. The
genetically related donor can include, but is not limited to, a mother,
father, sibling, grandparent, aunt, or uncle of the neonatal mammalian
subject. The exogenous brown adipocytes or the precursors thereof can be
derived from one or more of an autologous tissue, allogeneic tissue, or
xenogeneic tissue. The exogenous brown adipocytes or the precursors
thereof can be derived from a neonatal-associated tissue. The exogenous
brown adipocytes or the precursors thereof can be derived from one or
more of adipocytes, pre-adipocytes, stem cells, cord blood cells,
placental cells, myoblasts, or bone marrow cells. The method can further
include expanding, maturing, or differentiating the exogenous brown
adipocytes or the precursors thereof in vitro. The method can further
include providing one of more of differentiation factors or growth
factors in vitro to the exogenous brown adipocytes or the precursors
thereof. The brown adipocytes, or the precursors thereof further can
include one or more detectable markers incorporated with the brown
adipocytes or the precursors thereof. In some aspects, providing the
brown adipocytes or the precursors thereof to the internal site can
include injecting the brown adipocytes or the precursors thereof. The
method can further include injecting the brown adipocytes or the
precursors thereof in a pharmaceutically acceptable carrier. In some
aspects, providing the brown adipocytes or the precursors thereof to the
internal site can include implanting the brown adipocytes or the
precursors thereof. The method can further include providing the brown
adipocytes or the precursors thereof in a pharmaceutically acceptable
carrier. The method can further include providing the brown adipocytes or
the precursors thereof in one or more biocompatible carriers.

[0004] The method can further include encapsulating the brown adipocytes
or the precursors thereof. The method can further include providing the
brown adipocytes or the precursors thereof in an immunoisolating
material. The biocompatible carrier can include, but is not limited to,
at least one of a membrane, natural matrix, synthetic matrix, polymer,
scaffold, hydrogel, natural sponge, synthetic sponge, microbead,
microcapsule, microsphere, microparticle, or an encapsulating material.
The method can further include providing one or more medicaments for
modulating heat loss from the brown adipocytes. The one or more
medicaments can include, but is not limited to, one or more of a
neurotransmitter, a neurotrophic agent, a neuropeptide, an adipokine, or
an uncoupling protein. The one or more medicaments can include, but is
not limited to, one or more of a β3-adrenergic receptor agonist, NPY
antagonist, leptin, UCP activating agent, thyroxine, serotonin reuptake
inhibitor, MCH antagonist, GLP-1 agonist, 5-HT2C agonist, 5-HT2A agonist,
galanin antagonist, CRF agonist, urocortin agonist, melanocortin agonist
or enterostatin agonist.

[0005] A method is disclosed herein that includes: providing exogenous
brown adipocytes, or precursors thereof, to an internal site in a
neonatal mammalian subject. In some aspects, providing the brown
adipocytes can include providing pre-adipocytes to the neonatal mammalian
subject. The brown adipocytes, or the precursors thereof, can be
substantially purified. The method can further include providing factors
to the subject that promote at least one of adipocyte proliferation,
adipocyte differentiation, or adipose angiogenesis. The factors can
include, but are not limited to, differentiation factors, growth factors,
or angiogenic factors. The neonatal mammalian subject can include, but is
not limited to, a human, equine, bovine, ovine, swine, rodent, lagomorph,
canine, or feline. The neonatal mammalian subject can include a neonatal
mammalian subject born preterm.

[0006] In some aspects, modulating heat loss includes reducing heat loss
in the neonatal mammalian subject by providing the exogenous brown
adipocytes, or the precursors thereof, to the internal site in the
neonatal mammalian subject. The precursors can include, but are not
limited to, one or more of stem cells, totipotent stem cells, multipotent
stem cells, pluripotent stem cells, oligopotent stem cells, embryonic
stem cells, de-differentiated stem cells, trans-differentiated stem
cells, mesenchymal stem cells, adipose-derived stem cells, adipocyte
progenitor cells, pre-adipocytes, myoblasts, muscle-derived stem cells,
or bone marrow-derived stem cells. The exogenous brown adipocytes can
include mature brown adipocytes. The internal site can include, but is
not limited to, one or more of a subcutaneous site, scapular site,
axillary site, thoracic site, abdominal site, or blood vessel site of the
neonatal mammalian subject. The brown adipocytes or the precursors
thereof can be derived from one or more of cell donors, tissue donors,
tissue culture stock, cell lines, or genetically manipulated cells. The
genetically manipulated cells can include an exogenous DNA sequence
encoding one or more of a mammalian UCP polypeptide or a PRDM16
polypeptide.

[0007] The one or more cell donors or tissue donors can include a
genetically related donor of the neonatal mammalian subject. The
genetically related donor can include, but is not limited to, a mother,
father, sibling, grandparent, aunt, or uncle of the neonatal mammalian
subject. The exogenous brown adipocytes or the precursors thereof can be
derived from one or more of an autologous tissue, allogeneic tissue, or
xenogeneic tissue. The exogenous brown adipocytes or the precursors
thereof can be derived from a neonatal-associated tissue. The exogenous
brown adipocytes or the precursors thereof can be derived from one or
more of adipocytes, pre-adipocytes, stem cells, cord blood cells,
placental cells, myoblasts, or bone marrow cells. The method can further
include expanding, maturing, or differentiating the exogenous brown
adipocytes or the precursors thereof in vitro. The method can further
include providing one of more of differentiation factors or growth
factors in vitro to the exogenous brown adipocytes or the precursors
thereof. The brown adipocytes, or the precursors thereof further can
include one or more detectable markers incorporated with the brown
adipocytes or the precursors thereof. In some aspects, providing the
brown adipocytes or the precursors thereof to the internal site can
include injecting the brown adipocytes or the precursors thereof. The
method can further include injecting the brown adipocytes or the
precursors thereof in a pharmaceutically acceptable carrier. In some
aspects, providing the brown adipocytes or the precursors thereof to the
internal site can include implanting the brown adipocytes or the
precursors thereof. The method can further include providing the brown
adipocytes or the precursors thereof in a pharmaceutically acceptable
carrier. The method can further include providing the brown adipocytes or
the precursors thereof in one or more biocompatible carriers.

[0008] The method can further include encapsulating the brown adipocytes
or the precursors thereof. The method can further include providing the
brown adipocytes or the precursors thereof in an immunoisolating
material. The biocompatible carrier can include, but is not limited to,
at least one of a membrane, natural matrix, synthetic matrix, polymer,
scaffold, hydrogel, natural sponge, synthetic sponge, microbead,
microcapsule, microsphere, microparticle, or an encapsulating material.
The method can further include providing one or more medicaments for
modulating heat loss from the brown adipocytes. The one or more
medicaments can include, but is not limited to, one or more of a
neurotransmitter, a neurotrophic agent, a neuropeptide, an adipokine, or
an uncoupling protein. The one or more medicaments can include, but is
not limited to, one or more of a β3-adrenergic receptor agonist, NPY
antagonist, leptin, UCP activating agent, thyroxine, serotonin reuptake
inhibitor, MCH antagonist, GLP-1 agonist, 5-HT2C agonist, 5-HT2A agonist,
galanin antagonist, CRF agonist, urocortin agonist, melanocortin agonist
or enterostatin agonist.

[0009] The foregoing summary is illustrative only and is not intended to
be in any way limiting. In addition to the illustrative aspects,
embodiments, and features described above, further aspects, embodiments,
and features will become apparent by reference to the drawings and the
following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0010] FIGS. 1A and 1B are schematics of a diagrammatic view of an aspect
of an embodiment of a method for modulating heat loss in a neonatal
mammalian subject.

[0011] FIGS. 2A and 2B are schematics of a diagrammatic view of an aspect
of an embodiment of a method for modulating heat loss in a neonatal
mammalian subject.

[0012]FIG. 3 is a schematic of a diagrammatic view of an aspect of an
embodiment of a method for modulating heat loss in a neonatal mammalian
subject.

DETAILED DESCRIPTION

[0013] In the following detailed description, reference is made to the
accompanying drawings, which form a part hereof. In the drawings, similar
symbols typically identify similar components, unless context dictates
otherwise. The illustrative embodiments described in the detailed
description, drawings, and claims are not meant to be limiting. Other
embodiments may be utilized, and other changes may be made, without
departing from the spirit or scope of the subject matter presented here.

[0014] This document uses formal outline headings for clarity of
presentation. However, it is to be understood that the outline headings
are for presentation purposes, and that different types of subject matter
may be discussed throughout the application (e.g., method(s) may be
described under composition heading(s) and/or kit headings, and/or
descriptions of single topics may span two or more topic headings).
Hence, the use of the formal outline headings is not intended to be in
any way limiting.

[0015] Methods are disclosed herein for modulating heat loss in a neonatal
mammalian subject. The method includes providing exogenous brown
adipocytes, or precursors thereof, to an internal site, e.g., in vivo, in
the neonatal mammalian subject. In the method disclosed herein, providing
brown adipocytes can include providing pre-adipocytes or providing
precursor cells of exogenous brown adipocytes to an internal site in the
neonatal mammalian subject. The method can further include providing
factors that promote at least one of adipocyte proliferation, adipocyte
differentiation, or adipose tissue angiogenesis. The factors can include,
but are not limited to, differentiation factors, growth factors, or
angiogenic factors. Promoting at least one of adipocyte proliferation,
adipocyte differentiation, or adipose tissue angiogenesis can include,
but is not limited to, inducing, supporting, or enhancing at least one of
adipocyte proliferation, adipocyte differentiation, or adipose
angiogenesis. The factors that promote proliferation can be formulated to
induce support, or enhance differentiation of the pre-adipocytes or
adipocyte precursors to brown adipocytes. The internal site for placement
of brown adipocytes in the neonatal mammalian subject can include one or
more of a subcutaneous site, scapular site, axillary site, thoracic site,
abdominal site, or blood vessel site of the neonatal mammalian subject.
The internal site for placement of brown adipocytes in the neonatal
mammalian subject can further include regions of the neck, regions of the
back including back regions along the spine and above the buttock, and
regions of the scalp of the neonatal mammalian subject. In some aspects,
the brown adipocytes or precursors thereof can be substantially purified.

[0016] Methods are disclosed herein for modulating heat loss in a neonatal
mammalian subject. Modulating heat loss in the neonatal mammalian subject
can include, but is not limited to, decreasing the rate of heat loss or
the amount of heat loss in the neonatal mammalian subject, or controlling
a decrease or an increase in the rate of heat loss or the amount of heat
loss in the neonatal mammalian subject. The method includes providing
exogenous brown adipocytes, or precursors thereof, to an internal site,
e.g., in vivo, in the neonatal mammalian subject. In the method disclosed
herein, providing exogenous brown adipocytes can include providing
exogenous pre-adipocytes or providing exogenous precursor cells or
exogenous progenitor cells of brown adipocytes to an internal site in the
neonatal mammalian subject. The precursors can include, but are not
limited to, stem cells, totipotent stem cells, multipotent stem cells,
pluripotent stem cells, oligopotent stem cells, embryonic stem cells,
de-differentiated stem cells, trans-differentiated stem cells,
mesenchymal stem cells, adipose-derived stem cells, adipocyte progenitor
cells, pre-adipocytes, myoblasts, muscle-derived stem cells, or bone
marrow-derived stem cells. Precursors of brown adipocytes include, but
are not limited to, stem cells or progenitor cells that are in any stage
of development or differentiation. Stem cells include, but are not
limited to, cells that can proliferate and differentiate through
developmental stages into mature cell types. Progenitor cells include
cell descendants of stem cells that are committed to a developmental
lineage. Pre-adipocyte includes a specific progenitor cell of a brown
adipocyte or a white adipocyte.

[0017] The method can further include providing factors that promote
proliferation of brown adipocytes or precursors thereof. The method can
further include providing factors that promote or induce adipocyte
differentiation. The method can further include providing factors that
promote angiogenesis of adipose tissue. Factors provided can include, but
are not limited to, differentiation factors, growth factors, or
angiogenic factors. The differentiation factors, growth factors, or
angiogenic factors can be provided in vitro to cells in culture prior to
providing the cells to the neonatal mammalian subject. Alternatively, the
differentiation factors, growth factors, or angiogenic factors can be
provided in vivo with the exogenous cells at an internal site in the
neonatal mammalian subject. Various growth or angiogenic factors, e.g.,
basic fibroblast growth factor (bFGF), acidic fibroblast growth factor,
and vascular endothelial growth factor (VEGF) can also be administered as
an adjunct to improve vascularization of the tissue comprising the brown
adipocyte transplant. The differentiation factors, growth factors, or
angiogenic factors can be provided to the same site as the exogenous
cells in the neonatal mammalian subject, prior to, at the same time, or
after a time interval from providing the exogenous cells. The
differentiation factors, growth factors, or angiogenic factors can be
provided to a different site in the neonatal mammalian subject than the
site to which the exogenous cells have been provided. The factors can be
provided prior to, at the same time, or after a time interval from
providing the exogenous cells. The factors can be provided to the
neonatal mammalian subject systemically prior to, at the same time, or
after a time interval from providing the exogenous cells. The internal
site for placement of brown adipocytes in the neonatal mammalian subject
can include one or more in vivo sites in the neonatal mammalian subject
including one or more of a subcutaneous site, scapular site, axillary
site, thoracic site, abdominal site, or blood vessel site of the neonatal
mammalian subject. In some aspects, the brown adipocytes, or precursors
thereof, can be substantially purified.

[0018] The neonatal mammalian subject can include, but is not limited to,
a subject that is human, equine, bovine, ovine, swine, rodent, lagomorph,
canine, or feline. The non-human neonatal mammalian subjects can be used,
for example, in areas of husbandry, companion animals, purebred show
animals, or laboratory animals.

[0019] A method is described herein for modulating heat loss in a neonatal
mammalian subject by providing exogenous thermogenic brown adipocytes to
an internal site in the neonatal subject. In general, mammalian neonates
sustain life within a narrow range of core body temperature; for humans
this is generally from about 36.5° C. to about 37.5° C. The
mammalian body uses a number of mechanisms to balance heat loss and heat
production in order to maintain the core temperature within this range
despite a wide range of environmental temperatures. Balancing heat loss
and gain is known as thermoregulation. The range of ambient temperature
that maintains a mammalian neonate's core body temperature using minimum
oxygen consumption at a minimum metabolic rate is called the
thermoneutral range. See, e.g., Asakura, J. Nippon Med. Sch. 71: 360-370,
2004; which is incorporated herein by reference.

[0020] At birth, a human neonate's thermoregulatory mechanisms are not
fully developed, but the thermoregulatory mechanisms become progressively
more efficient during early development. Neonates exhibit limited
response to temperature changes in the first 24 hours of life and are
particularly susceptible to chilling during this time. By 24-48 hours of
age, healthy term neonates are able to increase their heat production up
to 2.5 times in response to cold; however, they remain susceptible to
environmental temperature changes. Until the mechanisms controlling
thermoregulation (and heat loss in particular) stabilize, the
thermoneutral range of infants is at a higher environmental temperature
than older children and adults. The method, as described herein, for
modulating heat loss in a neonatal mammalian subject by providing
exogenous thermogenic brown adipocytes to an internal site in the
neonatal subject can be used to enhance thermoregulatory mechanisms in
the first weeks of life of a human neonate.

[0021] Heat is easily lost at birth, when autonomic thermoregulation is at
its least efficient. Hypothermia (e.g., a core temperature of less than
36.5° C. in humans) can occur rapidly unless active steps are
taken to prevent heat loss, and perinatal hypothermia is a contributing
factor to adverse outcomes. The World Health Organization classifies a
newborn human core body temperature of 36.0 to 36.4 C° as mild
hypothermia, 32 to 35.9 C° as moderate hypothermia, and lower than
32 C° as severe hypothermia. The signs, symptoms, and
complications of hypothermia in a neonate can include, but are not
limited to, body cool to touch, mottling or pallor, central cyanosis,
acrocyanosis, poor feeding, abdominal distension, hypotonia,
hypoglycemia, increased gastric residuals, bradycardia, tachypnea,
restlessness, shallow or irregular respiration, apnea, and lethargy. See,
e.g., Bhatt, et al., J. Perinatology 27: S45-S47, 2007, which is
incorporated herein by reference.

[0022] A number of features contribute to ease of heat loss in neonates,
including a large surface area-to-mass ratio (in humans approximately
three times greater than adults); less subcutaneous fat; thin epidermis;
blood vessels closer to the skin allowing changes in ambient temperature
to more readily affect the circulating blood, thereby influencing the
temperature-regulating center in the hypothalamus; and a decreased
ability to shiver. In contrast, several features contribute to heat
production in full-term neonates including a decreased ability to sweat,
a flexed posture which conserves heat by reducing exposed surface area,
and the ability to generate heat by non-shivering thermogenesis. In
preterm neonates, these features are not fully developed, making it even
more difficult for the preterm neonates to maintain a normal body
temperature.

[0023] Non-shivering thermogenesis is facilitated by increased metabolic
activity in brown adipose tissue (BAT) resulting in the generation of
heat. Brown adipose tissue is so-named because it has a darker hue than
white adipose tissue due to a more extensive blood supply and greater
numbers of mitochondria. In response to cooling, signals are sent from
the central nervous system to the brown adipose tissue to induce
lipolysis, the enzymatic breakdown of trigylcerides to fatty acids.
Lipolysis in most cell types results in the generation of ATP. However,
in brown adipocytes, lipolysis is uncoupled from the production of ATP,
releasing the energy as heat. Uncoupling protein 1 (UCP-1) plays a
central role in facilitating the uncoupling process and the release of
heat. Oxygen is required for lipolysis, and lipolysis in brown adipose
tissue uses up to three times as much oxygen as other tissue. See, e.g.,
Nedergaard & Cannon, Cell Metab. 11 (4): 268-272, 2010; Cannon &
Nedergaard, "Brown adipose tissue: Function and physiological
significance," Physiol. Rev. 84:277-359, 2004; Morrison & Nakamura,
Frontiers in Bioscience 16: 74-104, 2011; Bartness, et al., International
Journal of Obesity 34: S36-S42, 2010; Lee, et al., Am J Physiol
Endocrinol Metab 299: E601-E606, 2010; Saely, et al., "Brown versus White
Adipose Tissue: A Mini-Review," Gerontology, Karger AG, Basel, Dec. 7,
2010; Bartell et al., Nat Med (2011) January 23, doi: 10.1038/nm.2297; Ma
et al., PLoS ONE 6 (1): e16391. doi:10.1371/journal.pone.0016391. 2011;
Richard and Picard, Frontiers in Bioscience 16: 1233-1260, 2011; which
are incorporated herein by reference.

[0024] BAT is thought to constitute 2-7% of total body weight in human
neonatal subjects. BAT is deposited in the fetus beginning at 26 weeks of
gestation, steadily increasing in amount until two to five week after
birth. At term, BAT is deposited around the nape of the neck and
mid-scapular area, under the clavicles and in the axillae, around the
kidneys, adrenal glands and large vessels in the neck and in the
mediastinum. See, e.g., Richard and Picard, Frontiers in Bioscience 16:
1233-1260, 2011; Heaton, J. Anat., 112: 35-39, 1972, which are
incorporated herein by reference. Maternal prostaglandins and adenosine
prevent non-shivering thermogenesis in utero; BAT is activated after
birth.

[0025] Preterm neonates and small-for-gestational-age neonates have less
brown adipose tissue and a greater surface area to mass ratio than
full-term neonates. Furthermore, the lower the gestational age, the less
brown adipose tissue is available for thermoregulation. In addition, the
muscles of preterm neonates are less developed, resulting in less flexing
of the body and limbs. Consequently, these infants are extremely
vulnerable to hypothermia. In general, maintaining body temperature is an
integral part of treatment in the preterm neonate and is extremely
challenging. Low temperature on admission to the neonatal intensive care
is an independent risk factor for mortality in extremely preterm
neonates. See, e.g, Costeloe, et al., Pediatrics 106: 659-671 2000; Lyon,
et al., Arch. Dis. Child. 76: F47-F50, 1997, which are incorporated
herein by reference. The method, as described herein, for modulating heat
loss in a neonatal mammalian subject by providing exogenous thermogenic
brown adipocytes to an internal site in the neonatal subject can be used
as part of a treatment for hypothermia in preterm neonates.

[0026] Low birth weight in human neonates and in nonhuman neonatal
mammals, e.g., domesticated animals, is a condition that indicates less
insulation for the neonatal mammal and also indicates a limited supply of
tissue substrates available as a source for energy conversion. The
method, as described herein, for modulating heat loss in a low birth
weight neonatal mammalian subject by providing exogenous thermogenic
brown adipocytes to an internal site in the neonatal mammalian subject
can be used alone or in combination with administration of nutritional
supplements such as glucose to the neonatal mammalian subject. The single
treatment or the combination treatment can be used to treat low birth
weight in preterm neonates or in full term neonates.

[0027] The method, as described herein, for modulating heat loss in a low
birth weight neonatal mammalian subject by providing exogenous
thermogenic brown adipocytes to an internal site in the neonatal
mammalian subject can be used to prevent starvation in nonhuman neonatal
mammalian subjects, e.g., when the neonate has not being accepted for
nursing by its mother. The method, as described herein, can be used to
treat conditions in human or nonhuman mammalian neonatal subjects
including, but not limited to, dystocia (difficult birth); in utero
exposure to drugs or alcohol, e.g., fetal alcohol syndrome, resulting in
small-for-gestational-age birth and/or hypothermia; or neonatal recurrent
prolonged hypothermia associated with maternal mirtazapine treatment
during pregnancy. See, e.g., Can J Clin Pharmacol 15 (2): e188-e190,
2008, which is incorporated herein by reference. Fetal alcohol syndrome
can have associated hypothermia and hypoglycemia.

[0028] With reference to the figures, and with reference now to FIGS. 1,
2, and 3, depicted is an aspect of a method that can serve as an
illustrative embodiment of and/or for subject matter technologies, for
example a method for modulating heat loss in a neonatal mammalian subject
that includes providing brown adipocytes, or precursors thereof, to an
internal site in the neonatal mammalian subject. The specific methods
disclosed herein are intended as merely illustrative of their more
general counterparts.

[0029] Referring to FIG. 1A, depicted is a partial diagrammatic view of a
method 100A for modulating heat loss in a neonatal mammalian subject that
includes providing exogenous brown adipocytes 110 to an internal site 120
in the neonatal mammalian subject 130. Brown adipocytes 110, including
adipocyte precursors and mature brown adipocytes, harvested from a
supraclavicular region of an allogeneic donor 140, e.g., a genetically
related donor such as the mother of the neonatal human subject, can be
transplanted to an internal site 120 in the neonatal subject 130. In some
aspects, the brown adipocytes 110, or the precursor cells or the
progenitor cells thereof, from the allogeneic donor 140 can be
substantially purified. The internal site 120 for injection of the brown
adipocytes into the neonatal subject can be an interscapular region of
the neonatal subject 130. The interscapular region for injection of the
brown adipocytes into the neonatal subject may already include some brown
adipose tissue.

[0030] Referring to FIG. 1B, depicted is a partial diagrammatic view of a
method 100B for modulating heat loss in a neonatal mammalian subject 130
that includes providing brown adipocytes 110, or precursors or
progenitors thereof 150, to an internal site 120 in the neonatal
mammalian subject 130. Adipose tissue including pre-adipocytes 150 can be
harvested from a subcutaneous region of an allogeneic donor 140, e.g., a
genetically related donor such as the mother of the neonatal human
subject. Cells from the adipose tissue including pre-adipocytes or
multi-potent adipose-derived stem cells 150 can be cultured in vitro in
the presence of growth factors to expand the cell number, and/or, e.g.
subsequently, in the presence of differentiation factors 160 to induce
differentiation into brown adipocytes 110. In some aspects, the brown
adipocytes 110, or the precursor cells or the progenitor cells thereof,
from the in vitro tissue culture can be substantially purified. The in
vitro cultured brown adipocytes 110 can be transplanted to an internal
site 120 in the neonatal subject 130. The internal site 120 of
transplantation of the brown adipocytes into the neonatal subject can be
an interscapular region of the neonatal subject 130.

[0031] Referring to FIG. 2A, depicted is a partial diagrammatic view of a
method 200A for modulating heat loss in a neonatal mammalian subject 230
that includes providing brown adipocytes 210, or precursors or
progenitors thereof 250, to an internal site 220 in the neonatal
mammalian subject 230. Umbilical cord blood including umbilical cord stem
cells 250 can be harvested from an umbilical cord, for example the
umbilical cord of a neonatal human subject collected at birth.
Alternatively, umbilical cord blood including umbilical cord stem cells
250 can be harvested from an umbilical cord of a donor 240, e.g., a
different neonatal mammal such as monozygotic or dizygotic twin of the
neonatal human subject, or an unrelated donor. For example, umbilical
cord stem cells 250 can be harvested from an umbilical cord of a donor
240, stored, and revived for use in the neonatal mammalian subject 230.
The umbilical cord blood including umbilical cord stem cells 250 can be
cultured in vitro in the presence of factors 260, for example growth
factors to expand the cell number and/or, e.g. subsequently,
differentiation factors to induce differentiation into brown adipocytes
210. In some aspects, the brown adipocytes 210, or the precursors
thereof, can be substantially purified from cells of umbilical cord blood
prior to or subsequent to culture. The in vitro cultured brown adipocytes
210 can be transplanted to an internal site 220 in the neonatal subject
230. The internal site 220 of transplantation of the brown adipocytes
into the neonatal subject can be an interscapular region of the neonatal
subject 230.

[0032] Referring to FIG. 2B, depicted is a partial diagrammatic view of a
method 200B for modulating heat loss in a neonatal mammalian subject 230
that includes providing brown adipocytes 210, or precursors or
progenitors thereof, to an internal site 220 in the neonatal mammalian
subject 230. Brown adipocytes 210 harvested from a supraclavicular region
of an allogeneic donor 240, e.g. a genetically related donor such as the
mother of the neonatal human subject, can be transplanted to an internal
site 220 in the neonatal subject 230. In some aspects, the brown
adipocytes 210, or the precursors or progenitors thereof, from the
allogeneic donor 240 can be substantially purified. The internal site 220
for transplantation of the brown adipocytes into the neonatal subject can
be an interscapular region of the neonatal subject 230. A medicament 270
formulated to modulate, e.g. induce, enhance, or control,
thermoregulation can be administered to the neonatal subject 230 in
combination with the transplanted brown adipose tissue 210.

[0033] Referring to FIG. 3, depicted is a partial diagrammatic view of a
method 300 for modulating heat loss in a neonatal mammalian subject 301
that includes providing exogenous brown adipocytes 310, or precursors
thereof, to an internal site in the neonatal mammalian subject. The
method can include providing brown adipocytes 310 or precursors thereof
and providing in vitro or in vivo, e.g., prior to, simultaneously, or
subsequently, exogenous factors 320, 330, 340 with the brown adipocytes
in the neonatal mammalian subject. The method can include expanding,
maturing, or differentiating 320 the exogenous brown adipocytes or
precursors thereof in vitro. The method can include providing growth
factors in vitro 330 formulated to expand cell number of brown adipocytes
or precursors thereof or providing differentiation factors in vitro 340
formulated to induce brown adipocyte differentiation. The method can
include expanding, maturing, or differentiating 350 the exogenous brown
adipocytes or precursors thereof in vivo. The method can include
providing growth factors in vivo 355 formulated to expand cell number of
brown adipocytes or precursors thereof or providing differentiation
factors in vivo 360 formulated to induce brown adipocyte differentiation.
The method can further include providing to the neonatal mammalian
subject angiogenic factors in vivo 365 formulated to promote tissue
vascularization.

[0034] The method for modulating heat loss can include reducing heat loss
in the neonatal mammalian subject by providing the exogenous brown
adipocytes, or the precursors thereof, to the internal site in the
neonatal mammalian subject. The method can include injecting 370 the
brown adipocytes or the precursors thereof to the internal site. The
method can include implanting 375 the brown adipocytes or the precursors
thereof to the internal site. The method can include providing 380 the
brown adipocytes or the precursors thereof in a pharmaceutically
acceptable carrier. The method can include providing 385 the brown
adipocytes or the precursors thereof in one or more biocompatible
carriers which can include encapsulating 390 the brown adipocytes or the
precursors thereof or providing 395 the brown adipocytes or the
precursors thereof in an immunoisolating material.

[0036] Exogenous brown adipocytes for transplantation into a neonatal
subject can be derived from brown adipose tissue isolated from a donor.
In some aspects, the brown adipocytes, e.g., mature brown adipocytes or
adipose-derived stem cells or progenitor cells, can be substantially
purified. The brown adipose tissue can be isolated from autologous tissue
or from an allogeneic or xenogeneic tissue donor. The donor can be
genetically related, either closely, e.g., a biological mother, father,
and/or sibling, or more distantly, e.g., a grandparent, aunt, and/or
uncle, or can be an unrelated individual. In some instances, the brown
adipose tissue can be derived from a xenogeneic tissue donor.

[0037] Brown adipose tissue in small mammals is located primarily in the
interscapular region and the axillae (i.e., underarm) and to a lesser
degree near the thymus and in the dorsal midline region of the thorax and
abdomen. A similar distribution of brown adipose tissue is observed in
full-term human neonates. In adult mammals, e.g., adult humans, depots of
functional brown adipose tissue are located in a region extending from
the anterior neck to the thorax, and primarily in the supraclavicular
region, as well as the cervical and mediastinal regions. Additional
depots are found associated with blood vessels and some internal organs,
e.g, kidneys. See, e.g., Virtanen, et al., N. Engl. J. Med., 360:
1518-1525, 2009; Cypess, et al., N. Engl. J. Med., 360: 1509-1517, 2009;
and van Marken Lichtenberg et al., N. Engl. J. Med., 360: 1500-1508,
2009, which are incorporated herein by reference. Brown adipose tissue
for transplantation can be resected from a donor by biopsy, surgery, or
liposuction. See, e.g., U.S. Patent Application No. 2010/0015104, which
is incorporated herein by reference.

[0038] Stem cell refers to a cell having the capacity to self-renew and to
differentiate into mature, specialized cells. A totipotent stem cell can
differentiate into any type of body cell in addition to all of the
extraembryonic cell types. A pluripotent stem cell can differentiate into
any type of body cell. A multipotent stem cell can differentiate into
multiple cell types of a particular tissue, organ, or physiological
system. A progenitor cell is a stem cell descendent that can
differentiate to give rise to a cell in a distinct lineage. In
particular, brown adipocyte progenitor cell, e.g., a pre-adipocyte or an
early progenitor cell, refers to a cell with the potential to
differentiate into brown adipocytes.

[0039] Brown adipocytes for transplantation into a neonatal subject can be
derived from differentiation of pre-adipocytes. Pre-adipocytes are
progenitor cells committed to the adipocyte lineage. Exposure of
pre-adipocytes to differentiation factors results in morphological and
biochemical changes including cell rounding and accumulation of
triacylglycerol and lipid vacuoles. Pre-adipocytes can be isolated from
adipose tissue removed from a subject or donor by biopsy, surgery or
aspiration. In an aspect, the pre-adipocytes can be obtained from the
subcutaneous fat of the neonate subject for autologous cell
transplantation. Alternatively, the pre-adipocytes can be obtained from
an appropriate donor for allogeneic cell transplantation. The
pre-adipocytes can be obtained from a genetically related donor, e.g., a
biological mother, father, or sibling, and prepared for transplantation
either prior to or shortly after the birth of the neonatal subject. The
pre-adipocytes can be isolated from adipose tissue after disruption and
digestion, e.g., with one or more enzymes. Immunoselection and/or
depletion can be used to further select for CD34.sup.+/CD31.sup.pre-adipocytes. See, e.g., Elabd, et al., Stem Cells 27: 2753-2760, 2009;
Gomillion & Burg, Biomaterials 27: 6052-6063, 2006, which are
incorporated herein by reference. Pre-adipocytes are also available from
commercial sources (e.g., from Zen-Bio, Research Triangle Park, N.C.;
Genlantis, San Diego, Calif.).

[0040] Pre-adipocytes isolated from white adipose tissue or brown adipose
tissue can be cultured in differentiation medium containing one or more
of insulin, isobutyl methylxanthine (IBMX), dexamethasone, and
transferrin. Pre-adipocytes can be differentiated into brown adipocytes
in the presence of a PPARγ (peroxisome proliferator-activated
receptor γ) selective agonist, e.g., rosiglitazone. See, e.g.,
Elabd, et al., Stem Cells 27: 2753-2760, 2009, which is incorporated
herein by reference. Pre-adipocytes derived from subcutaneous white
adipose tissue or brown adipose tissue can in addition or alternatively
be cultured in the presence of one or more bone morphogenetic protein
(BMP), such as BMP2, BMP4, BMP6 or BMP7, which can promote
differentiation into brown adipocytes; in particular BMP7 can be added to
the culture medium to promote differentiation into brown adipocytes.
(See, e.g., Tseng et al., Nature 454 (7207): 1000-1004, 2008, which is
hereby incorporated by reference). Pre-adipocytes derived from
subcutaneous white adipose tissue can also be differentiated into brown
adipocytes by transfecting the pre-adipocytes with a coactivator of
PPARγ called PPARγ coactivator 1α (PGC-1α). See,
e.g., Tiraby, et al., J. Biol. Chem., 278: 33370-33376, 2003, which is
incorporated herein by reference. Adenovirus-mediated expression of
PGC-1α increases the expression of UCP1, respiratory chain
proteins, and fatty acid oxidation enzymes in human subcutaneous white
adipocytes, while other changes in expression are consistent with that of
brown adipocyte mRNA expression profile. In some instances, brown
adipocytes can be derived from pre-adipocytes isolated from brown adipose
tissue and immortalized using a viral promoter, e.g., SV40 T oncogene
promoter. See, e.g., U.S. Pat. No. 6,071,747, which is incorporated
herein by reference.

[0041] Brown adipocytes for transplantation into a neonatal subject can be
derived from differentiation of stem cells or progenitor cells. Stem
cells can include, but are not limited to, embryonic stem cells
(including cells from embryonic stem cell lines), adult stem cells,
induced pluripotent stem cells, mesenchymal stem cells, totipotent stem
cells, multipotent stem cells, pluripotent stem cells, adipose-derived
stem cells, muscle-derived stem cells, or bone-marrow derived stem cells.
In general a stem cell refers to a cell having the capacity to self-renew
and to differentiate into mature, specialized cells. A totipotent stem
cell can differentiate into any type of body cell plus all of the
extraembryonic cell types. A pluripotent stem cell can differentiate into
to any type of body cell, and a multipotent stem cell can differentiate
into multiple cell types of a particular tissue, organ, or physiological
system. An example of pluripotent stem cells includes, but is not limited
to, embryonic stem cells. An example of multipotent stem cells includes
hematopoietic stem cells that can differentiate into any blood cell type.
Mesenchymal stem cells are multipotent stem cells that can be
differentiated into a variety of cell types, including adipocytes.
Mesenchymal cells can be isolated from adipose tissue as well as from
bone marrow, skeletal muscle, umbilical cord blood, placenta, and others.
See, e.g., WO2006/051538; Elabd, et al., Stem Cells 27: 2753-2760, 2009;
Lee, et al., Blood, 103:1669-1675, 2004; and Gomillion & Burg,
Biomaterials, 27: 6052-6063, 2006; which are incorporated herein by
reference.

[0043] Exogenous brown adipocytes for transplantation into a neonatal
mammalian subject can be derived from stem cells or progenitor cells
isolated from adipose or nonadipose tissue isolated from an autologous
tissue or from allogeneic donor tissue. Tissue or cell sources from which
to derive stem cells or progenitor cells that are capable of developing
into mature adipocytes include, but are not limited to, bone marrow,
muscle tissue, adipose tissue, umbilical tissue, embryonic stem cells,
adult stem cells, or stem cell lines. For example, bone marrow contains
mesenchymal stem cells, which can be differentiated into adipocytes. Bone
marrow can be isolated by aspiration, and a density gradient can be used
to isolate a small percentage of mesenchymal stem cells. These cells will
proliferate until exposed to culture medium containing differentiation
factors. Adipogenic differentiation of mesenchymal cell cultures derived
from bone marrow can be induced to form adipocytes by treatment with
isobutyl methylxanthine, dexamethasone, insulin, and indomethacin. See,
e.g., Pittenger, et al., Science, 284: 143-147, 1999, which is
incorporated herein by reference. In some protocols for differentiation
of mesenchymal stem cells isolated from adipose, following growth to
confluence, 20 nM to 100 nM rosiglitazone is added to culture medium and
dexamethasone and isobutyl methylxanthine are omitted. See, e.g., Elabd,
et al., Stem Cells 27: 2753-2760, 2009, which is incorporated herein by
reference. Similar methods have been described for generating adipocytes
from progenitor cells isolated from adipose, cord-blood, placenta, or
skeletal muscle. See, e.g., Kang, et al., Cell Biol. Int. 30: 569-575,
2006; Park, et al., Cell Metab., 8: 454-457, 2008; Seale, et al., Nature,
454: 961-968, 2008; and WO2006/051538, which are incorporated herein by
reference.

[0044] Brown adipocytes for transplantation into a neonatal mammalian
subject can be derived from stem cells or from myoblasts isolated from
fetal, neonatal, or mature skeletal muscle. Brown adipocytes and myocytes
appear to share a common precursor, specifically a cell lineage that
expresses the Myf5 gene. See, e.g., Seale, et al., Genes Dev. 23:
788-797, 2009, which is incorporated herein by reference. Brown
adipocytes for use in a neonatal subject can be derived from CD34.sup.+
(CD146.sup.-, CD45.sup.-, & CD56.sup.-) progenitor cells isolated by
digestion and immunoselection/depletion from skeletal muscle. See, e.g.,
Crisan, et al., Stem Cells, 26: 2425-2433, 2008, which is incorporated
herein by reference. Brown adipocytes for transplantation can also be
derived from skeletal muscle myoblast cell lines. For example, ectopic
expression of PRDM16 in primary or established committed myoblasts causes
the cells to adopt a brown adipocyte phenotype when exposed to
pro-adipogenic stimuli. See, e.g., Seale, et al., Nature 454: 961-967,
2008, which is incorporated herein by reference. Brown adipocytes for use
in a neonatal subject can be derived from multipotent stem cells or
myoblasts isolated from enzyme-dissociated muscle tissue. See, e.g.,
Asakura et al., Differentiation 68: 245-253, 2001, which is incorporated
herein by reference. Myoblasts for use in differentiation into brown
adipocytes for use in transplantation into human neonatal subjects can be
can be derived from biopsy or resection of skeletal muscle. See, e.g.,
Baj, et al., J. Translational Med. 3 (21): 2005 and De Coppi, et al.,
Diabetologia, 49 (8): 1962-73, 2006, which are incorporated herein by
reference. In some instances, myoblast cell lines are available from a
commercial source, examples of which include, but are not limited to,
C2C12, H9c2(2-1), G-7, L6, A-10, L8, So18, and LHCN-M2 (e.g., from
American Type Culture Collection (ATCC), Manassas, Va.).

[0047] Pre-adipocytes, precursors, stem cells, or progenitor cells,
including those isolated from the neonatal subject-associated tissue,
allogeneic donor-associated tissue, or established or primary cell lines
can be expanded, and appropriate differentiation factors added to induce
differentiation of the cells into brown adipocytes. The stem cells can be
administered to the neonatal subject at any cell stage and can be matured
or differentiated either in vitro or in vivo. The differentiation of
pre-adipocytes or progenitor stem cells into brown adipocytes can include
stimulation by one or more growth factors, differentiation factors, or
transcriptional factors. In general, pre-adipocytes or progenitor cells,
or stem cells are differentiated into adipocytes by exposure to
pro-adipogenic stimuli. In vitro, the pro-adipogenic stimulus can be a
cocktail of reagents added to the cell culture medium and can include one
or more of 3-isobutyl-1-methylxanthine, dexamethasone, dibutyryl cyclic
AMP, insulin, transferrin, triodothyronine, rosiglitazone, and
indomethacin. Factors can also be used to promote differentiation of the
adipocyte lineage. For example, one or more bone morphogenetic protein
(BMP), such as BMP2, BMP4, BMP6 or BMP7 can be added to the culture to
promote differentiation into brown adipocytes. In particular BMP7 can be
added to the culture medium. When BMP7 is added in culture to brown
pre-adipocytes isolated from adipose tissue, the expression of UCP-1
increases, as does the expression of several regulators of adipocyte
differentiation including PRDM16, PGC-1α, and PGC-1β. In
addition, BMP7 induces an increase in mitochondrial density, consistent
with the phenotype of differentiated brown adipocytes. Similarly, the
addition of BMP7 to multipotent stem cells, e.g., C3H10T1/2 cells,
results in a mature brown adipocyte phenotype with marked increases in
lipid accumulation and induction of UCP-1. See, e.g., U.S. Pat. No.
7,576,052 and Tseng, et al., Nature, 454: 1000-1004, 2008, which are
incorporated herein by reference.

[0048] In general, the nuclear hormone receptor peroxisome
proliferator-activated receptor γ (PPARγ) appears to be
important for development of brown adipose tissue. For example,
PPARγ-deficient mice (PPARγ-/-) do not develop normal brown
adipose tissue (BAT) deposits and die shortly at or before gestational
day 12.5. The activity of PPARγ can be stimulated by a variety of
ligands including, but not limited to, natural eicosanoid
15-deoxy-Δ.sup.12,14-prostaglandin J2, endogenous constituents
of oxidized low density lipoprotein (oxLDL) particles, e.g., 9- and
13-HODE, perfluorononanioc acid, and synthetic thiazolidinedione (TZD)
compounds, e.g., rosiglitazone, pioglitazone, rivoglitazone, and
ciglitazone. Including one or more PPARγ agonist in the culture
medium can aide differentiation of precursor cells into adipocytes. See,
e.g., Barak, et al., Molecular Cell, 4: 585-595, 1999, which is
incorporated herein by reference.

[0049] The differentiation of pre-adipocytes, precursors, progenitor
cells, or stem cells into brown adipocytes can also be stimulated by
treatment with one or more transcription factors. Examples of
transcription factors involved in adipocyte differentiation include, but
are not limited to, RBI, p107, RBL1, RIP 140, NRIP1, FOXC2, PRDM16,
GATA2. For example, PRDM16 ("PR domain containing 16") is a
transcriptional co-regulatory protein and contributes to brown adipocyte
differentiation by co-activating PPARy, the latter of which is considered
the master gene of adipocyte differentiation. PRDM16 can also complex
with other DNA-binding factors, e.g., C/ERP-β to initiate brown
adipocyte differentiation. See, e.g., Seale et al., Genes Dev., 23:
788-797, 2009; Cannon & Nedergaard, Physiol. Rev., 84: 277-359, 2004; and
U.S. Patent Application 2011/0059051, which are incorporated herein by
reference.

[0050] Pre-adipocytes, precursors, progenitor cells, or stem cells can be
differentiated into brown adipocytes in vitro in the presence of a
pro-adipogenic reagent mix, components of which can include, but are not
limited to isobutyl methylxanthine, dexamethasone, dibutyryl cyclic AMP,
insulin, transferrin, triodothyronine, rosiglitazone, indomethacin and
ascorbic acid. When the cells have reached an appropriate cell number and
confluency (e.g., 70-95% confluence), a pro-adipogenic reagent mix is
added to induce terminal differentiation of the cells into brown
adipocytes. After about 3 to 30 days in pro-adipogenic reagent mix, the
cells can be assessed for phenotypes characteristic of brown adipocytes
using one or more assays. Assays for assessing phenotypes characteristic
of brown adipocytes include, but are not limited to, staining with
Oil-red O to detect lipid droplets, polymerase chain reaction (PCR)
amplification, Northern blot analysis, and/or Western blot analysis to
detect expression of brown adipocyte specific transcripts and proteins
(e.g., UCP-1), and microscopy to detect increased mitochondrial numbers.
See, e.g., U.S. Application 2009/0054487 and PCT Application
WO/2005/123049A2, which are incorporated herein by reference.

[0051] Differentiation of pre-adipocytes, progenitor cells, or stem cells
into brown adipocytes can be facilitated by genetically modifying the
cells to express factors known to promote brown adipocyte
differentiation. Gene transfer techniques are known by persons of
ordinary skill in the art, and can include viral and non-viral
transfection techniques. See, e.g., Verma, et al., Gene Therapy 5:
692-699, 1998; and Papapetrou, et al., Gene Therapy 12: S118-S130, 2005,
which are incorporated herein by reference. Inducing PRDM 16 gene
expression in adipocytes can induce brown adipocyte differentiation in
the mammalian subject. Increasedbrown fat differentiation in the
mammalian subject can induce the expression of mitochondrial genes and
cellular respiration leading to a reduction in obesity in the mammalian
subject. See, e.g., U.S. Patent Application 2011/0059051; PCT WO
2005/123049A2, which are incorporated herein by reference. For example,
forced expression of the zinc-finger protein PRDM16 or the transcription
factor C/EBP-13 in stromal-vascular stem cells isolated from white
adipose tissue or in naive fibroblastic cells using retroviral gene
transfer vectors induces a brown adipocyte phenotype as exemplified by
upregulation of UCP-1 and other markers of brown adipocyte
differentiation including PGC1α, Cox8b, elov13, Adipoq, and
Adipsin. Transplanting cells modified with PRDM16 and C/EBP-β into a
mammalian subject results in depots of functional brown adipose tissue as
exemplified by a positive response to 18F-fluorodeoxyglucose
computed tomography. Ectopic over-expression of UCP-1 in pre-adipocytes
has also been described. See, e.g., Seale, et al., Cell Metab., 6: 38-54,
2007; and Kajimura, et al., Nature 460: 1154-1158, 2009; Si, et al., J.
Lipid Res., 48: 826-838, 2007, which are incorporated herein by
reference.

[0052] The brown adipocytes for transplantation into a neonatal subject
can be modified to include genetic modifications that change, enhance, or
supplement the function of the transplanted cells. The genetic
modifications can include expression of gene products that promote
differentiation of the pre-adipocytes, precursors, progenitor cells, or
stem cells into metabolically active brown adipocytes. The genetic
modifications can include expression of gene products that can promote
the vascularization of the tissue comprising the transplanted brown
adipocytes. Examples of gene products that can promote the
vascularization of the tissue comprising the transplanted brown
adipocytes include VEGF and other known angiogenic factors. Similarly,
the genetic modifications can include expression of modulators of brown
adipocyte function. Examples of modulators of brown adipocyte function
include BMP7 or activators of β adrenergic receptors. In an aspect,
genetic modification to change, enhance, or supplement the function of
the transplanted brown adipocytes can be made to non-adipocytes that are
configured to express growth and/or differentiation factors and are
co-transplanted with the brown adipocytes.

[0053] The brown adipocytes for transplantation into a neonatal mammalian
subject can be modified to include a marker to enable tracking of the
cells following transplantation to determine viability and sustainability
of the transplanted cells. The marker can be one or more of a fluorescent
marker, a magnetic marker, an RFID tag, a radioactive marker, a
radiopaque marker or a combination thereof that can be used to monitor
the transplanted cells. In some instances, the marker can be genetically
incorporated into the brown adipocytes, for example, a green fluorescent
protein marker. More generally, the increase in brown adipocytes
represented by the transplanted cells can be reflected in increased
glucose metabolism as monitored by positron-emission tomography (PET) and
computed tomography (CT) in the presence of 18F-fluorodeoxyglucose
as described by Virtanen, et al., (in N. Engl. J. Med. 360: 1518-1525,
2009, which is incorporated herein by reference). Alternatively, the
activity of brown adipose tissue in neonates can be assessed using
gamma-camera imaging with 99mTc-tetrofosmin. See, e.g., Fukuchi, et
al., J. Nucl. Med., 44: 1582-1585, 2003, which is incorporated herein by
reference.

[0054] The brown adipocytes for transplantation into a neonatal mammalian
subject can be identified and further regulated by regulating expression
of uncoupling protein 1 (UCP1) in the brown adipocytes. The brown
adipocytes for transplantation into a neonatal mammalian subject can be
identified by expression of UCP1 as a marker. UCP1 is an important
component of non-shivering thermogenesis mediated by brown adipose
tissue. UCP1 is also a specific marker of differentiation of
pre-adipocytes and progenitor cells into brown adipocytes. UCP1 is a 32
kDa protein expressed in the inner membrane of the mitochondria. UCP1
allows the dissipation of the proton electrochemical gradient generated
by the respiratory chain. Uncoupling between oxygen consumption and ATP
synthesis promotes energy dissipation as heat. Fatty acids and retinoids
have been shown to activate UCP1. In neonatal mammals, hibernators and
rodents, cold-induced thermogenesis in brown adipose tissue (BAT)
contributes to the maintenance of body temperature. Fuel is provided as
fatty acids derived from brown adipocyte and white adipocyte lipolysis.
UCP1 biosynthesis is mainly controlled at the transcriptional level.
During cold exposure, for example, sympathetic nervous system stimulation
of brown adipose tissue is the primary signal that activates UCP1 gene
expression.

Methods For Administration of A Therapeutic Composition Including Brown
Adipocytes

[0056] A method for modulating heat loss in a neonatal mammalian subject
is described herein that includes providing exogenous brown adipocytes,
or precursors thereof, to an internal site in the neonatal mammalian
subject. The method further includes providing the brown adipocytes to an
internal site including, but not limited to, a subcutaneous site, a
scapular site, an axillary site, a thoracic site, an abdominal site, or
blood vessel site of the neonatal mammalian subject. The brown adipocytes
can be provided to a neonatal mammalian subject by implantation and/or by
injection. Implantation can be achieved by injection.

[0057] The brown adipocytes can be provided to a neonatal mammalian
subject by injection, for example, by injecting into a subcutaneous site,
an intraperitoneal site, intramuscular site, or vascular site. The brown
adipocytes can be administered to a neonatal mammalian subject, e.g., by
injection, as a cell suspension in a pharmaceutically acceptable carrier.
In an aspect, the pharmaceutically acceptable carrier is a suspension
fluid, for example, normal saline (0.91% w/v sodium chloride). Other
suitable suspension fluids for cells or tissues include, but are not
limited to, lactated Ringer's solution or serum-free culture medium with
or without additives including, but not limited to, heparin, insulin,
vitamin E, and nonsteroidal anabolic hormones. The suspension fluid can
further include one or more of the medicaments described herein to
promote the growth and viability of the brown adipocytes or surrounding
tissue or affect function thereof.

[0059] The brown adipocytes can be provided to a neonatal mammalian
subject e.g. by implantation and/or by injection in a formulation that
includes a hydrogel. Hydrogels can be used as a growth matrix for
pre-adipocytes, progenitor cells, stem cells, and/or partially or fully
differentiated brown adipocytes. Cell culture-incorporating and/or
cell-encapsulating hydrogels can be used as injectable cell carriers.
Examples of hydrogels for use providing brown adipocytes include, but are
not limited to, alginate, hyaluronic acid, polyethylene glycol (PEG),
and/or fibrin gels. The hydrogel gelation can be controlled by
temperature or chemical means: In an aspect, brown adipocytes can be
combined with a hydrogel just prior to injection, in a form encapsulating
the cells, as described herein. Brown adipocytes can be homogenously
mixed in a hydrogel in its liquid state and subsequently injected as a
gel state. The brown adipocytes can be homogenously mixed in a
temperature-sensitive hydrogel in its liquid state at room temperature
(22-25° C.) and allowed to gel upon reaching body temperature
(e.g., 37° C., after subcutaneous injection). Alternatively or in
addition, brown adipocytes and their precursors can be cultured on
hydrogels, and can be expanded and/or induced to differentiate prior to
injection.

[0060] Certain hydrogels, including some available from commercial
sources, are derivatives of extracellular and/or basement membrane
products. BD Matrigel® Matrix and BD® Laminin/Entactin Complex are
available from Becton Dickinson have been used for culturing human
embryonic stem cells and induced pluripotent stem cells, and can
potentiate in vivo adipogenesis of exogenously applied pre-adipocytes in
mammals. Myogel, available from Biolink in Australia, is derived from an
muscle tissue, and adipose protein-derived and dermis-derived gels that
can sustain adipogenesis have also been established and investigated in
vivo. Fibrin hydrogels encapsulating adipose-derived stem cells have been
successfully implanted in mammals into both subcutaneous and
intramuscular tissues. Polyethylene glycol (PEG) is a synthetic hydrogel
commonly used for tissue engineering. Stem cells, including cells
adipogenically induced, have successfully been implanted in mammalian
subcutaneous tissues when encapsulated in PEG-di(meth)acrylate (PEGDA)
hydrogel. These synthetic hydrogels can be modified with peptides to
promote cell adhesion. See Bauer-Kreisel, et al., Cell-delivery
therapeutics for adipose tissue regeneration, Advanced Drug Delivery
Reviews 62: 798-813, 2010.

[0061] A scaffold, whether natural or synthetic, refers to a
three-dimensional template for colonization by pre-adipocytes, progenitor
cells, stem cells, and/or partially or fully differentiated brown
adipocytes. Colonization can take place in vitro, e.g. in a cell culture,
or in vivo, e.g., when the scaffold is implanted and exogenous cells
provided separately. In an aspect, the scaffold can include one or more
biodegradable polymers. Examples of natural polymers for use in a
three-dimensional matrix include, but are not limited to, polypeptides,
e.g., albumin, fibrinogen, fibrin, collagen and gelatin, hyaluronic acid,
and also polysaccharides, e.g., chitin, chitosan, alginate and agarose.
These natural polymers can also be modified, where appropriate; for
example, proteins such as collagen can be crosslinked. Examples of
synthetic polymers for use in a three-dimensional matrix include, but are
not limited to, polyanhydrides, e.g., poly(sebacic acid-hexadecanoic
diacid), poly(ε-caprolactone), poly(orthoesters),
(polytetrafluoroethylene), polyethylene glycol (PEG), and, especially,
poly(α-hydroxy-esters), e.g., poly(glycolic acid), poly(lactic
acid), poly(glycolic acid-lactic acid) (PGA/PLA/PLGA). Scaffolds can be
designed in a variety of geometric shapes and with defined physical
properties including porosity, diameter and surface area.

[0062] In an aspect, a scaffold can be designed to be soft and non-rigid
with optimum surface area, for example, using a natural material to form
a sponge having a certain size pore or a synthetic polymer forming a high
surface area fiber. Pre-adipocytes, precursors, progenitor cells, stem
cells, and/or partially or fully differentiated brown adipocytes can be
seeded onto a soft biocompatible scaffold, with or without encapsulation,
and can be provided to a neonatal mammalian subject, e.g. by implantation
in a subcutaneous site. The biocompatible scaffold can be seeded and
colonized ex vivo prior to implantation and/or can be implanted/injected
into the neonatal subject and seeded in situ. For example, a collagen or
gelatin sponge, e.g. having a pore size of 200-400 micrometers, may be
seeded with pre-adipocytes, which are then expanded and differentiated in
vitro. The gelatin sponges carrying the cells can be implanted into
subcutaneous tissue of the abdomen of the neonatal mammalian subject
following a small incision. A porous gelatin sponge is available
commercially (Gelfoam, Pharmacia & Upjohn, Kalamazoo, Mich., USA) and has
been used extensively in clinical and tissue engineering studies as a
scaffold. Adipose-derived stem cells grown on the gelatin sponge, and
induced to differentiate, then subcutaneously implanted in an adult
mammal. See, e.g., Hong et al., Adipose Tissue Engineering by Human
Adipose-Derived Stromal Cells, Cells Tissues Organs, 183: 133-140, 2006.

[0064] The brown adipocytes can be provided to an internal site of the
neonatal mammalian subject, e.g. by implantation and/or by injection, in
a formulation that includes microbeads, microcapsules, microparticles, or
microspheres. Cells can be adhered to or cultured on solid or porous
microspheres or microparticles that can then be provided to the neonatal
subject, either alone or in combination with a hydrogel. For example,
cells can be precultured on poly(lactic-co-glycolic) acid (PLGA)
microspheres as the cell carrier, e.g., including adipogenic induction,
and still have the carrier in an injectable form. See, e.g., P.
Bauer-Kreisel et al. Advanced Drug Delivery Reviews 62: 798-813, 2010.
For example, microparticles constructed from crosslinked chitosan can be
used as an.sub.:injectable platform for tissue engineering. See, e.g.,
Cruz, et al., Chitosan microparticles as injectable scaffolds for tissue
engineering, J. Tissue Eng. Regen. Med. 2: 378-380, 2008, which is
incorporated herein by reference. Microparticles or microspheres can be
generated using one or more of the natural or synthetic polymers
described herein, examples of which include, but are not limited to,
albumin, fibrinogen, collagen, gelatin, chitin, chitosan, alginate,
agarose, poly(sebacic acid-hexadecanoic diacid),
poly(ε-caprolactone), poly(orthoesters), poly(glycolic acid),
poly(lactic acid) or poly(glycolic acid-lactic acid), or may be derived
from tissue, including extracellular matrix powders. Microspheres or
microparticles may also be modified to enhance better cell adhesion; for
example, alginate beads can be modified with the cell adhesion peptide
RGD.

[0065] The brown adipocytes can be provided to an internal site of the
neonatal mammalian subject, e.g., by implantation, including surgical
implantation into, e.g., a subcutaneous site, intramuscular site,
intraperitoneal site or vascular site as a three dimensional tissue. For
example, brown adipocytes from human adipose-derived stem cells can be
cultured as a three dimensional sheets employing a "self-assembly"
culture methodology, then harvested and provided to the neonatal human
subject by subcutaneous implantation. See, e.g., P. Bauer-Kreisel et al.
Advanced Drug Delivery Reviews 62: 798-813, 2010.

[0066] The matrix, scaffold, or microparticle/microsphere material can be
designed to include chemical or surface modifications that enhance
attachment, proliferation, differentiation, and/or sustainability of the
brown adipocytes. For example, the material can include one or more
adhesive molecules to promote cellular attachment and/or one or more
growth factors or other medicaments described herein that promotes the
growth or viability of the brown adipocytes. Examples of adhesive
molecules to which cells are known to attach include one or more
extracellular matrix proteins, e.g., collagen, laminin and fibronectin,
as well as adherence-promoting peptides such as RGD. Examples of
biologically active substances for surface modification of a natural or
synthetic matrix, scaffold, or microparticle include, but are not limited
to, synthetic active compounds (inorganic or organic molecules),
proteins, polysaccharides and other sugars, lipids and nucleic acids
which, for example, influence cell growth, cell migration, cell division,
cell differentiation and/or tissue growth or possess therapeutic,
prophylactic or diagnostic effects. Examples of biologically active
substances for surface modification of a natural or synthetic matrix,
scaffold, or microparticle can further include, but are not limited to,
vasoactive active compounds, neuroactive active compounds, hormones,
growth factors, cytokines, steroids, anticoagulants, anti-inflammatory
active compounds, immunomodulating active compounds, cytotoxic active
compounds, antibiotics and antiviral active compounds. The matrix,
scaffold, or microparticle/microsphere material can include one or more
factors for promoting growth, e.g., FGF. The material can include one or
more factors for inducing differentiation, e.g., insulin and
dexamethasone. See, e.g., Rubin, et al., Encapsulation of adipogenic
factors to promote differentiation of adipose-derived stem cells, J Drug
Target. 17 (3): 207-215, 2009 which is incorporated herein by reference

[0068] Methods for modulating heat loss in a neonatal mammalian subject
can include providing brown adipocytes for transplantation to an internal
site of the neonatal mammalian subject. The brown adipocytes can be
delivered, for example, by implantation and/or injection, as encapsulated
brown adipocytes. In an aspect, encapsulation can provide immunoisolation
of the brown adipocytes in the neonatal subject to prevent an immune
response and rejection of the transplanted cells. Encapsulation can be
particularly advantageous when the brown adipocytes are derived from an
allogeneic or xenogeneic source. Immunoisolation can be achieved by
encapsulating the cells, including the scaffolds or matrix on which they
are grown, in a semi-permeable, biocompatible material that isolates the
transplanted cells from attack by the neonatal subject's immune system
but allows for exchange of nutrients, gases, and signaling molecules
between the transplanted cells and the neonatal mammalian subject.

[0069] A variety of semipermeable polymeric and inorganic matrices and
membranes with diverse physiochemical properties and geometries can be
used to encapsulate the brown adipocytes and provide immune-isolation in
the neonatal mammalian subject. Examples of encapsulating materials can
include one or more membranes, natural matrix, synthetic matrix,
macroparticles, microparticles, microspheres, macrocapsules, or
microcapsules as described herein.

[0070] In an aspect, the brown adipocytes can be encapsulated in a natural
matrix or synthetic matrix including an alginate. Alginates are a family
of unbranched anionic polysaccharides originally derived from brown
algae. Alginates are a linear polysaccharide copolymers with
homopolymeric and heteropolymeric blocks of (1-4)-linked
β-D-mannuronate (M) and α-L-guluronate (G) residues linked
together in different sequences or blocks. In the presence of divalent
cations, e.g., calcium, the alginate forms a biocompatible gelatin-like
material stable at body temperature into which cells for transplantation
can be encapsulated. See, e.g., U.S. Pat. Nos. 5,084,350 and 5,429,821;
Ghidoni, et al., Cytotechnol. 58: 49-56, 2008, which are incorporated
herein by reference. Encapsulation in alginate has been demonstrated to
provide protection from an immune response for allogeneic or xenogenic
transplantation of cells in a number of mammalian species including
humans. See, e.g., Lim, et al., Cell microencapsulation, Adv Exp Med
Biol. 670: 126-136, 2010, which is incorporated herein by reference.
Encapsulation of stem cells in alginate, and specifically adipose-derived
stem cells, has been described. See, e.g., Liu and Chang, Artificial cell
microencapsulated stem cells in regenerative medicine, tissue engineering
and cell therapy, Adv Exp Med BioL 670: 68-79, 2010 and U.S. Pat. No.
7,078,232, which are incorporated herein by reference.

[0072] In an aspect, immunoisolation of the brown adipocytes from the
neonatal immune system can be achieved using an immunoisolation system
that includes a cage formed from a natural material such as one or more
protein or lipid, or a synthetic material such as hydroxyapatite. An
example includes structures composed of natural or synthetic bone-like
material, e.g., hydroxyapatite, configured to provide living cells and
tissues to subjects in a rigid or semi-rigid conformation. See, e.g.,
U.S. Patent Application 2008/0057095, which is incorporated herein by
reference.

Promotion of Angiogenesis In Brown Adipose Tissue

[0073] A method for modulating heat loss in a neonatal mammalian subject
is described herein that includes providing exogenous brown adipocytes,
or precursors thereof, to an internal site in the neonatal mammalian
subject. The method can further include providing factors to the subject
that promote adipose angiogenesis. Angiogenesis refers to the process by
which new blood vessels are generated from existing vasculature and
tissue. See, e.g., Folkman, J., Nat Med 1: 27-31, 1995, which is
incorporated herein by reference. "Repair or remodeling" refers to the
reformation of existing vasculature. The spontaneous growth of new blood
vessels provides collateral circulation in and around an area of
implanted brown adipocytes, improves blood flow, and alleviates the
symptoms caused by ischemia. Angiogenic factor or angiogenic protein
refers to any protein, peptide or other agent capable of promoting growth
of new blood vessels from existing vasculature ("angiogenesis"). Suitable
angiogenic factors for use to induce vascularization of brown adipose
tissue include, but are not limited to, placenta growth factor,
macrophage colony stimulating factor, granulocyte macrophage colony
stimulating factor, vascular endothelial growth factor (VEGF)-A, VEGF-A,
VEGF-B, VEGF-C, VEGF-D, VEGF-E, neuropilin, fibroblast growth factor
(FGF)-1, FGF-2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6, angiopoietin 1,
angiopoietin 2, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-β, IGF-1,
osteopontin, pleiotropin, activin, endothelin-1, and combinations
thereof. Angiogenic factors can act independently, or in combination with
one another. When in combination, angiogenic factors can also act
synergistically, whereby the combined effect of the factors is greater
than the sum of the effects of the individual factors taken separately.

[0074] Angiogenic factors can be produced or obtained from any suitable
source. For example, the factors can be purified from their native
sources, or produced synthetically or by recombinant expression.
Angiogenic factors are commercially available. The factors can be
provided to the neonatal subject together with the brown adipocytes. For
example, the factors can be provided with the cells in an injectable
formulation. The factors may be provided with the cells as part of the
growth matrix or scaffold, e.g., in a slow release formulation or as part
of the modified matrix or scaffold material. The factors may be provided
during culture, expansion, and/or differentiation. Instead or in
addition, the factors can be administered to the neonatal subject
directly, e.g. as a medicinal composition. Alternatively, the factors can
be administered in the form of an expression plasmid encoding the
factors. The construction of suitable expression plasmids is well known
in the art. Suitable vectors for constructing expression plasmids
include, for example, adenoviral vectors, retroviral vectors,
adeno-associated viral vectors, RNA vectors, liposomes, cationic lipids,
lentiviral vectors and transposons. See, e.g., U.S. Pat. No. 7,651,684
and U.S. Application 2010/00015104, which are incorporated herein by
reference.

[0075] Alternatively or in addition, vascular structures can be pre-formed
in vitro, for example in three dimensional cultures of adipocytes grown
on scaffolds. For three-dimensional constructs of substantially large
size, an early vascular supply throughout the whole composite can be a
crucial requirement for adequate and homogeneous tissue development and
long-term survival. Brown adipocytes cultured on scaffolds can include
preformation of a capillary network by in vitro addition of endothelial
cells and proangiogenic factors or addition of other engineered vascular
constructs. See, e.g., Bauer-Kreisel et al. Advanced Drug Delivery
Reviews 62: 798-813, 2010.

Internal Sites For Transplantation of Brown Adipocytes

[0076] A method for modulating heat loss in a neonatal mammalian subject
further includes providing the brown adipocytes to an internal site in
the neonatal mammalian subject, including, but not limited to, a
subcutaneous site, a subdermal site, a scapular site, an axillary site, a
thoracic site, an abdominal site, or blood vessel site of the neonatal
mammalian subject. Subcutaneous administration refers to administration
to an area of the mammalian anatomy located beneath the skin or beneath
the dermis. Administration to a scapular site refers to administration to
an area of the mammalian anatomy proximal to the scapula or shoulder
blade. A scapular site can further include an interscapular region or
subscapular region. Administration to an interscapular region refers to
administration to an area of the mammalian anatomy between the scapulae
on the upper back. Administration to a subscapular region refers to
administration to an area of the mammalian anatomy situated below or on
the underside of the scapula. Administration to an axillary site refers
to administration to an area of the mammalian anatomy under the arm,
e.g., in the area of the armpit. Administration to a thoracic site refers
to administration to an area of the mammalian anatomy pertaining to the
thorax or that region of the body that lies between the head and the
abdomen and includes the thoracic cavity containing the heart and lungs.
Administration to an abdominal site refers to administration to an area
of the mammalian anatomy that lies between the thorax and the pelvis and
includes the abdominal cavity containing organs of the digestive and
urinary systems. Administration to a blood vessel site refers to
administration to an area of the mammalian anatomy that is either in or
proximal to a blood vessel or a capillary bed. Administration to an
internal site can further include administration to the supraclavicular
region which refers to an area of the mammalian anatomy situated above
the clavicle. Administration can include implantation or injection;
implantation may be achieved by injection. Administration may be
described as transplantation or implantation.

[0079] Examples of medicaments that can be used to modulate heat loss in
the neonatal mammalian subject and to modulate the thermogenic activity
of brown adipocytes and reduce heat loss in the neonatal subject include
exogenous thyroid-active substances that include, but are not limited to,
T4 hormone and its analogs (L-thyroxine, levothyroxine,
3,5,3',5'-tetraiodo-L-thyronine), T3 hormone and its analogs
(3,5,3'-triiodo-L-thyronine, liothyronine, tertroxin, cytomel), T3/T4
combinations (liotrix, Thyrolar®), thyroid hormone precursors
(thyroglobulin, proloid), thyroprotein, thyroactive iodinated casein,
thyroid hormone, dessicated thyroid extracts (thyroidine). Substances
that stimulate the thyroid to produce T4 or T3 can be administered, that
include, but are not limited to, TSH (thyroid-stimulating hormone,
thyrotropin, thyrotropic hormone, Ambinon® or Dermathycin®), and
TRH (thyrotropin-releasing hormone). See, e.g., U.S. Patent Application
2006/0008512; U.S. Patent Application 2002/0160394, which are
incorporated herein by reference.

[0080] Medicaments can further include growth factors or other agents to
promote the viability, proliferation, expansion, and/or differentiation
of the transplanted brown adipocytes, or to promote vascularization
and/or neuralization of the tissue comprising the transplanted brown
adipocytes or precursors thereof. Examples of factors that positively
affect adipose growth or differentiation include, but are not limited to,
glucocorticoid, growth hormone, IGF-1, insulin, prostaglandins, or
thyroid hormone, and other examples provided herein. Examples of
angiogenic or arteriogenic growth factors that promote or stabilize
vascularization include, but are not limited to, VEGF, FGF,
angiopoietins, matrix metalloproteinase, Delta-like ligand 4, PDGF,
TGF-β, plasminogen activators, or eNOS. See, e.g., Patrick, Anat.
Rec. 263: 361-366, 2001, which is incorporated herein by reference.
Examples of factors that promote neural outgrowth or stabilization
include, but are not limited to, NGF, erythropoietin, as well as a number
of the factors listed above.

[0081] Under conditions in which the transplanted brown adipocytes are
from an allogeneic or xenogeneic origin, the medicaments can include one
or more immunosuppressive agents to reduce and/or prevent rejection of
the transplanted brown adipocytes. An immunosuppressive agent can be one
or more of an agent of drug which inhibits or interferes with normal
immune function. Examples of immunosuppressive agents that inhibit T-cell
and/or B-cell costimulation pathways include, but are not limited to,
cyclosporine A, tacrolimus, mycophenolate mofetil, rapamycin, or
anti-thymocyte antibodies.

[0082] In general, medicaments can be administered on any of a number of
dosing schedules depending upon the nature of the medicament. The
medicament can be administered simultaneously with the injected brown
adipocytes, e.g., in the same syringe, by separate local administration
at the injection site, or by systemic administration. The medicament can
be administered before, during and/or after injection of the brown
adipocytes. The duration of treatment can be, e.g., approximately 1-6
days, approximately 1-4 weeks, or approximately 1-6 months. In an aspect,
administration of the medicament can be discontinued once the neonatal
subject achieves normal levels of thermoregulation. In the case of an
immunosuppressive agent, the medicament may need to be administered on a
regular basis over the lifetime of the transplanted cells which in some
instances may be until the neonatal mammalian subject can thermoregulate
normally without assistance from the transplanted brown adipocytes.

[0083] A method is described for modulating heat loss in a neonatal human
subject. The method includes transplanting allogeneic mature brown
adipose tissue provided by a related donor to a subcutaneous site in the
recipient neonatal subject. Mature brown adipose tissue is harvested from
the related donor, who is the mother of the neonatal human subject. The
mature brown adipose tissue is transplanted to a subcutaneous site, e.g.,
the interscapular region, of the neonatal subject.

[0084] Mature brown adipose tissue is harvested from the mother of the
neonatal human subject using liposuction. Depots of brown adipose tissue
are located in the supraclavicular region of the mother using
positron-emission tomography (PET) and computed tomography (CT) at
ambient temperature of 17° C. in the presence of
18F-fluorodeoxyglucose. See, e.g., Virtanen, et al., N. Engl. J. Med.
360: 1518-1525, 2009, which is incorporated herein by reference. A local
anesthetic, e.g., lidocaine, is used to anesthetize the site of harvest.
An aspiration cannula connected to a syringe is inserted through the skin
into or near the identified brown adipose tissue depot in the
supraclavicular region of the mother of the neonatal human subject. The
brown adipose tissue is suctioned by hand or with a mechanical vacuum.
Suction is applied slowly to avoid excessive negative pressure, which can
contribute to breakage and/or vaporization of the cells. A moderate
amount of adipose tissue is harvested (e.g., from about 0.1 milliliters
to about 50 milliliters of adipose tissue).

[0085] Once the aspirate of mature brown adipose tissue has been
harvested, it can be directly injected into the neonate or further
processed to remove maternal blood and other non-adipocyte components.
The aspirate is washed in sterile normal saline (0.91% w/v of NaCl,
˜300 mOsm/L) by gently transferring the cells/tissue through
multiple syringes using tulip connections to 1 mL tuberculin syringes.
The adipocytes are resuspended in sterile normal saline in preparation
for transplantation.

[0086] The resuspended brown adipocytes (approximately 1-2 million cells
per ml) are loaded into a syringe fitted with an 18 gauge needle. Prior
to subcutaneous injection of the brown adipocyte cell suspension at a
site in the interscapular region of the neonatal subject, the site is
anesthetized with a local anesthetic, for example, lidocaine. A single
subcutaneous injection or multiple subcutaneous injections at varied
sites in the interscapular region are performed, depending upon the
volume and number of cells to be transplanted.

[0087] Body temperature measurements are used to monitor response to the
transplanted brown adipose tissue in the neonatal human subject. The body
temperature of the neonatal subject is monitored on a regular basis,
e.g., every hour, using an axillary underarm thermometer (e.g., from
Exergen Corporation, West

[0088] Yorkshire, UK). Temperatures in the range of 36.5° C. to
37.5° C. are considered appropriate for preterm neonates.
Secondary measurements include monitoring for signs of hypothermia
including skin temperature and pallor, respiration and heart rate,
feeding behavior, and activity level. If the body temperature of the
neonatal subject is not attaining a thermoregulated state, additional
brown adipocytes may be administered to the internal site to increase
thermoregulation in the neonatal subject. If the body temperature of the
neonatal subject is not reaching thermoregulation, a possible cause may
be lack of viability or rejection of the tissue. The viability of the
transplanted cells is then monitored by positron-emission tomography
(PET) and computed tomography (CT) in the presence of
18F-fluorodeoxyglucose as described by Virtanen, et al., (in N.
Engl. J Med. 360:1518-1525, 2009, which is incorporated herein by
reference). An immune response and/or rejection of the transplanted
allogeneic cells can be monitored by assessing antibodies and other
immune-related molecules against the donor cells. To avoid a
donor-mediated immune response or graft versus host disease (GVHD), or to
protect the allogeneic cells from an immune response, the brown
adipocytes may be encapsulated within a non-immunogenic material. The
brown adipocytes may be encapsulated in a combination of alginate and
poly-L-lysine. See, e.g., Goren et al., FASEB Journal, 24: 22-31, 2010
and Machluf, et al., Endocrinology 144: 4975-4979, 2003, which are
incorporated herein by reference. The degree of vascularization of the
transplanted brown adipose can be monitored using Doppler ultrasound,
either with or without a microbubble contrast agent. See, e.g., Krix, et
al., Cancer Res. 63: 8264-8270, 2003, which is incorporated herein by
reference.

Example 2

Modulating Heat Loss In A Neonatal Human Subject By Administering
Allogeneic Pre-Adipocytes Cultured In Vitro In the Presence of
Differentiation Factors

[0089] A method is described for modulating heat loss in a neonatal human
subject. The method includes transplanting brown adipocytes to a
subcutaneous site in the neonatal subject. The brown adipocytes are
derived from in vitro differentiation of pre-adipocytes isolated from the
mother of the neonatal subject and differentiated into brown adipocytes.
Optimally, the isolation and differentiation of the mother's
pre-adipocytes into brown adipocytes is performed prior to the birth of
the neonatal human subject such that the cells are available for
transplantation immediately upon delivery of the neonate. The brown
adipocytes are encapsulated in alginate to provide immunoisolation of the
cells and to prevent rejection of the cells by the neonatal subject. The
encapsulated brown adipocytes derived from differentiation of
pre-adipocytes of the mother are administered by subcutaneous injection
into the interscapular region of the neonatal subject.

[0090] Pre-adipocytes for use in transplantation are isolated from
subcutaneous white adipose tissue (WAT) of the mother of the neonatal
subject. WAT from the mother is harvested using liposuction as described
above. The adipose tissue is digested with collagenase (300 U/ml in
phosphate buffered saline, 2% bovine serum albumin, pH 7.4) for 45
minutes under constant shaking. The mature white adipocytes are removed
and the stromal vascular fraction is centrifuged, treated with
erythrocyte lysis buffer (155 mM NH4Cl, 5.7 mM K2HPO4, 0.1
mM EDTA, pH 7.3) and passed through 100, 70, and 40 μm sieves.
Immunoselection/depletion is used to isolate CD34.sup.+/CD31.sup.- cells,
defined as pre-adipocyte progenitor cells. See, e.g., Elabd, et al., Stem
Cells 27: 2753-2760, 2009, which is incorporated herein by reference.

[0091] The pre-adipocytes are cultured in RegES xeno-free medium
supplemented with FGF to encourage proliferation and are grown to
confluency. See, e.g., Rajala, et al., PLoS One. 5 (4): e10246, 2010,
which is incorporated herein by reference. The expanded cell culture of
pre-adipocytes is then transferred to pro-adipogenic culture medium to
induce differentiation into brown adipocytes. The pro-adopogenic medium
includes a combination of 10 μg/ml transferrin, 0.85 μM insulin,
0.2 nM triiodothyronine, 1 μM dexamethasone, 500 μM isobutyl
methylxanthine (IBMX) and, optionally, 20-500 nM rosiglitazone. See,
e.g., Elabd, et al., Stem Cells 27: 2753-2760, 2009, which is
incorporated herein by reference. Over the course of 3 to 16 days, the
differentiating progenitor cells are monitored for the expression of
uncoupling protein-1 (UCP-1), a marker of brown adipocytes. UCP-1
expression is monitored by Western blot analysis with anti-UCP-1 specific
antibodies (e.g., from Sigma-Aldrich, St. Louis, Mo.) or by a polymerase
chain reaction (PCR) assay. A second marker of brown adipocytes is
carnitine palmitoyltransferase (CPT1B). The CPT1B marker may be monitored
by Western blot analysis or by polymerase chain reaction (PCR). The cells
are also monitored for the emergence of lipid droplets within the
cytoplasm as determined using Oil Red-O staining and light microscopy.

[0092] The differentiated brown adipocytes are further processed by
encapsulation in alginate to provide immunoisolation of the cells from
the immune system of the neonatal subject. The cells are encapsulated in
alginate essentially as described by Tsai, et al., Biomed. Eng. Appl.
Basis Comm. 18: 62-66, 2006, which is incorporated herein by reference.
The differentiated brown adipocytes (105cells/ml) are mixed with
sodium alginate (e.g., from Sigma-Aldrich, St. Louis, Mo.) in phosphate
buffered saline (PBS) and extruded from a syringe through a 23 gauge
cannula into a 1.5% CaCl2 solution to form spherical gel beads. The
gel beads are collected and washed, and further coated with an aqueous
solution of 0.1% poly(L-lysine) to stabilize the gel beads. The gel beads
are incubated in culture medium containing transferrin, insulin,
triiodothyronine, dexamethasone, and isobutyl methylxanthine as described
above until transplantation into the neonatal subject.

[0093] The encapsulated differentiated brown adipocytes are injected into
the interscapular area of the neonatal subject. The encapsulated brown
adipocytes (approximately 1-2 million cells per ml) are washed and
suspended in buffered saline, then loaded into a syringe fitted with an
18 gauge needle. Prior to subcutaneous injection of the brown adipocyte
cell suspension at a site in the interscapular region of the neonatal
subject, the site is anesthetized with a local anesthetic, for example,
lidocaine. A single subcutaneous injection or multiple subcutaneous
injections at varied sites in the interscapular region are done,
depending upon the volume and number of cells to be transplanted. The
viability and activity of the encapsulated brown adipocytes are monitored
at one to two week intervals using gamma-camera imaging with
99mTc-tetrofosmin as described by Fukuchi, et al., J. Nucl. Med.,
44: 1582-1585, 2003 which is incorporated herein by reference. For this
analysis, 99mTc-tetrofosmin is injected into a peripheral vein of
the neonatal subject and 45 minutes later scans are taken using the
gamma-camera. In certain patients, including those at risk for cardiac
problems, the metabolic activity of brown adipose tissue may also or
instead be measured by the uptake of 18F-fluorodeoxyglucose
(18F-FDG) and PET imaging. See, e.g., Lee, et al., Am J Physiol
Endocrinol Metab 299: E601-E606, 2010; Saito, et al., Diabetes
58:1526-1531, 2009; Yoneshiro et al., Obesity (2011) 19: 13-16, 2011; van
Marken Lichtenbelt, et al., N. Engl. J. Med., 360: 1500-1508, 2009; and
Virtanen, et al., N. Engl. J. Med. 360: 1518-1525, 2009, which are each
incorporated herein by reference.

[0094] Body temperature measurements are used to monitor response to the
encapsulated differentiated brown adipocytes. The body temperature of the
neonatal subject is monitored on a regular basis, e.g., every hour, using
an axillary underarm thermometer (e.g., from Exergen Corporation, West
Yorkshire, UK). Temperatures in the range of 36.5° C. to
37.5° C. are considered appropriate for preterm neonates.
Secondary measurements include monitoring for signs of hypothermia
including skin temperature and pallor, respiration and heart rate,
feeding behavior, and activity level. Additional tests will be considered
to assess viability and/or rejection of the transplants adipocytes as
described herein.

[0095] A method is described for modulating heat loss in a neonatal human
subject. The method includes transplanting autologous brown adipocytes
derived from in vitro differentiation of mesenchymal stem cells isolated
from tissue associated with the neonatal human subject. Mesenchymal stem
cells are isolated from the cord blood of the neonatal subject
immediately following delivery of the neonate. The mesenchymal stem cells
are differentiated into brown adipocytes.

[0097] The differentiated brown adipocytes are injected into the
interscapular region of the neonatal subject. The brown adipocytes
(approximately 1-2 million cells per ml) are washed and resuspended in
buffered saline, then loaded into a syringe fitted with an 18 gauge
needle. Prior to subcutaneous injection of the brown adipocyte suspension
at a site in the interscapular region of the neonatal subject, the site
is anesthetized with a local anesthetic, for example, lidocaine. A single
subcutaneous injection or multiple subcutaneous injections at varied
sites in the interscapular region are done, depending upon the volume and
number of cells to be transplanted.

[0098] Body temperature measurements are used to monitor response to the
encapsulated differentiated brown adipocytes. The body temperature of the
neonatal subject is monitored on a regular basis, e.g., every hour, using
an axillary underarm thermometer (e.g., from Exergen Corporation, West
Yorkshire, UK). Temperatures in the range of 36.5° C. to
37.5° C. are considered appropriate for preterm neonates.

[0099] Secondary measurements include monitoring for, signs of hypothermia
including skin temperature and pallor, respiration and heart rate,
feeding behavior, and activity level. Additional tests will be considered
to assess viability and/or rejection of the transplanted adipocytes as
described herein.

[0100] A method is described for modulating heat loss in a neonatal human
subject. The method includes transplanting allogeneic mature brown
adipose tissue provided by a donor. The allogeneic mature brown adipose
tissue is transplanted to a subcutaneous site in the neonatal subject.
Mature brown adipose tissue is harvested from the mother of the neonatal
human subject, and transplanted to a subcutaneous site at the nape of the
neck of the neonatal subject. A medicament to control thermoregulation is
administered in combination with the transplanted brown adipose tissue.
The medicament administered for controlling thermoregulation is
levothyroxine, a synthetic thyroid hormone. Thyroid hormone normally
surges following full-term delivery and is necessary for the onset of
independent thermoregulation. Rapid stimulation of thyroid hormone
secretion contributes in part to increased expression of uncoupling
protein 1 (UCP-1). In contrast, thyroid hormone levels in preterm infants
younger than 30 weeks' gestation are considerably lower than full term
infants. The postnatal surge of thyroid hormone levels normally observed
in the first 24-48 hours is not observed in the preterm infants. In
addition, the lower the hormone levels, the worse the prognosis for the
neonate as measured by death or ventilator dependence at two weeks of
age. See, e.g., Biswas, et al., Pediatrics, 109: 222-227, 2002; and Gate,
et al., Experimental Physiology 85: 439-444, 2000, which are incorporated
herein by reference.

[0101] Mature brown adipose tissue is harvested from the mother of the
neonatal human subject by liposuction as described herein. Depots of
brown adipose tissue are located in the supraclavicular region of the
mother using positron-emission tomography (PET) and computed tomography
(CT) at an ambient temperature of 17° C. in the presence of
18F-fluorodeoxyglucose as described by Virtanen, et al., N. Engl. J.
Med. 360: 1518-1525, 2009, which is incorporated herein by reference. The
mature brown adipocytes are prepared for transplantation as described
herein.

[0102] The brown adipocytes isolated from mature brown adipose tissue are
prepared in a manner so as to prevent an immune response by the immune
system of the neonatal subject. The brown adipocytes are encapsulated in
a combination of alginate and poly-L-lysine. See, e.g., Goren et al.,
FASEB Journal vol. 24 no. 1 22-31, 2010, and Machluf, et al.,
Endocrinology 144:4975-4979, 2003, which are incorporated herein by
reference. The isolated mature brown adipocytes are suspended in a
solution of sodium alginate and saline (1.2% weight/volume) to a final
ratio of approximately 1-3×106 cells per 1 ml of alginate. The
suspension is sprayed through a 22-gauge needle into a calcium chloride
solution. The resulting cell-calcium-alginate beads are washed several
times in buffered saline and then collated with a 0.1% solution of
poly-L-lysine. An additional coating of alginate can also be added.

[0103] The encapsulated brown adipocytes are injected into the
interscapular region of the neonatal subject. The encapsulated brown
adipocytes (approximately 1-2 million cells per ml) are washed and
resuspended in buffered saline, then loaded into a syringe fitted with an
18 gauge needle. Prior to subcutaneous injection of the brown adipocyte
cell suspension at a site in the interscapular region of the neonatal
subject, the site is anesthetized with a local anesthetic, for example,
lidocaine. A single subcutaneous injection or multiple subcutaneous
injections at varied sites in the interscapular region are done,
depending upon the volume and number of cells to be transplanted.

[0104] A medicament to control thermoregulation is administered in
combination with the transplanted brown adipose tissue. The medicament
for controlling thermoregulation is levothyroxine, a synthetic thyroid
hormone, which is administered in conjunction,with transplantation of the
mature brown adipose tissue. Levothyroxine sodium is administered to the
neonatal subject as recommended in the prescribing information available
from U.S. Food & Drug Administration (FDA). For neonates unable to
swallow tablets, the tablet of levothyroxine sodium is finely crushed and
resuspended in about 5 to 10 milliliters of water. The suspension is
administered to the neonatal subject by dropper. The recommended starting
dose of levothyroxine sodium for neonates is 10-15
micrograms/kilogram/day. Lower doses of levothryoxine can be considered
depending upon the level of endogenous thyroid hormone.

[0105] Each recited range includes all combinations and sub-combinations
of ranges, as well as specific numerals contained therein.

[0106] All publications and patent applications cited in this
specification are herein incorporated by reference to the extent not
inconsistent with the description herein and for all purposes as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference for all purposes.

[0107] Those having ordinary skill in the art will recognize that the
state of the art has progressed to the point where there is little
distinction left between hardware and software implementations of aspects
of systems; the use of hardware or software is generally (but not always,
in that in certain contexts the choice between hardware and software can
become significant) a design choice representing cost vs. efficiency
tradeoffs. Those having ordinary skill in the art will recognize that
there are various vehicles by which processes and/or systems and/or other
technologies disclosed herein can be effected (e.g., hardware, software,
and/or firmware), and that the preferred vehicle will vary with the
context in which the processes and/or systems and/or other technologies
are deployed. For example, if a surgeon determines that speed and
accuracy are paramount, the surgeon may opt for a mainly hardware and/or
firmware vehicle; alternatively, if flexibility is paramount, the
implementer may opt for a mainly software implementation; or, yet again
alternatively, the implementer may opt for some combination of hardware,
software, and/or firmware. Hence, there are several possible vehicles by
which the processes and/or devices and/or other technologies disclosed
herein may be effected, none of which is inherently superior to the other
in that any vehicle to be utilized is a choice dependent upon the context
in which the vehicle will be deployed and the specific concerns (e.g.,
speed, flexibility, or predictability) of the implementer, any of which
may vary. Those having ordinary skill in the art will recognize that
optical aspects of implementations will typically employ
optically-oriented hardware, software, and or firmware.

[0108] In a general sense the various aspects disclosed herein which can
be implemented, individually and/or collectively, by a wide range of
hardware, software, firmware, or any combination thereof can be viewed as
being composed of various types of "electrical circuitry." Consequently,
as used herein "electrical circuitry" includes, but is not limited to,
electrical circuitry having at least one discrete electrical circuit,
electrical circuitry having at least one integrated circuit, electrical
circuitry having at least one application specific integrated circuit,
electrical circuitry forming a general purpose computing device
configured by a computer program (e.g., a general purpose computer
configured by a computer program which at least partially carries out
processes and/or devices disclosed herein, or a microprocessor configured
by a computer program which at least partially carries out processes
and/or devices disclosed herein), electrical circuitry forming a memory
device (e.g., forms of random access memory), and/or electrical circuitry
forming a communications device (e.g., a modem, communications switch, or
optical-electrical equipment). The subject matter disclosed herein may be
implemented in an analog or digital fashion or some combination thereof.

[0109] The herein described components (e.g., steps), devices, and objects
and the description accompanying them are used as examples for the sake
of conceptual clarity and that various configuration modifications using
the disclosure provided herein are within the skill of those in the art.
Consequently, as used herein, the specific examples set forth and the
accompanying description are intended to be representative of their more
general classes. In general, use of any specific example herein is also
intended to be representative of its class, and the non-inclusion of such
specific components (e.g., steps), devices, and objects herein should not
be taken as indicating that limitation is desired.

[0110] With respect to the use of substantially any plural or singular
terms herein, the reader can translate from the plural to the singular or
from the singular to the plural as is appropriate to the context or
application. The various singular/plural permutations are not expressly
set forth herein for sake of clarity.

[0111] The herein described subject matter sometimes illustrates different
components contained within, or connected with, different other
components. It is to be understood that such depicted architectures are
merely examples, and that in fact many other architectures can be
implemented which achieve the same functionality. In a conceptual sense,
any arrangement of components to achieve the same functionality is
effectively "associated" such that the desired functionality is achieved.
Hence, any two components herein combined to achieve a particular
functionality can be seen as "associated with" each other such that the
desired functionality is achieved, irrespective of architectures or
intermedial components. Likewise, any two components so associated can
also be viewed as being "operably connected," or "operably coupled," to
each other to achieve the desired functionality, and any two components
capable of being so associated can also be viewed as being "operably
couplable," to each other to achieve the desired functionality. Specific
examples of operably couplable include but are not limited to physically
mateable or physically interacting components or wirelessly interactable
or wirelessly interacting components or logically interacting or
logically interactable components.

[0112] While particular aspects of the present subject matter described
herein have been shown and described, changes and modifications may be
made without departing from the subject matter described herein and its
broader aspects and, therefore, the appended claims are to encompass
within their scope all such changes and modifications as are within the
true spirit and scope of the subject matter described herein.
Furthermore, it is to be understood that the invention is defined by the
appended claims. It will be understood that, in general, terms used
herein, and especially in the appended claims (e.g., bodies of the
appended claims) are generally intended as "open" terms (e.g., the term
"including" should be interpreted as "including but not limited to," the
term "having" should be interpreted as "having at least," the term
"includes" should be interpreted as "includes but is not limited to,"
etc.). It will be further understood that if a specific number of an
introduced claim recitation is intended, such an intent will be
explicitly recited in the claim, and in the absence of such recitation no
such intent is present. For example, as an aid to understanding, the
following appended claims may contain usage of the introductory phrases
"at least one" and "one or more" to introduce claim recitations. However,
the use of such phrases should not be construed to imply that the
introduction of a claim recitation by the indefinite articles "a" or "an"
limits any particular claim containing such introduced claim recitation
to inventions containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least one"
and indefinite articles such as "a" or "an"; the same holds true for the
use of definite articles used to introduce claim recitations. In
addition, even if a specific number of an introduced claim recitation is
explicitly recited, such recitation should typically be interpreted to
mean at least the recited number (e.g., the bare recitation of "two
recitations," without other modifiers, typically means at least two
recitations, or two or more recitations). Furthermore, in those instances
where a convention analogous to "at least one of A, B, and C, etc." is
used, in general such a construction is intended in the sense one having
skill in the art would understand the convention (e.g., "a system having
at least one of A, B, and C" would include but not be limited to systems
that have A alone, B alone, C alone, A and B together, A and C together,
B and C together, or A, B, and C together, etc.). In those instances
where a convention analogous to "at least one of A, B, or C, etc." is
used, in general such a construction is intended in the sense one having
skill in the art would understand the convention (e.g., "a system having
at least one of A, B, or C" would include but not be limited to systems
that have A alone, B alone, C alone, A and B together, Aand C together, B
and C together, or A, B, and C together, etc.). Virtually any disjunctive
word and/or phrase presenting two or more alternative terms, whether in
the description, claims, or drawings, should be understood to contemplate
the possibilities of including one of the terms, either of the terms, or
both terms. For example, the phrase "A or B" will be understood to
include the possibilities of "A" or "B" or "A and B."

[0113] While various aspects and embodiments have been disclosed herein,
other aspects and embodiments will be apparent to those skilled in the
art. The various aspects and embodiments disclosed herein are for
purposes of illustration and are not intended to be limiting, with the
true scope and spirit being indicated by the following claims.

Patent applications by Edward K.y. Jung, Bellevue, WA US

Patent applications by Edward S. Boyden, Chestnut Hill, MA US

Patent applications by Elizabeth A. Sweeney, Seattle, WA US

Patent applications by Eric C. Leuthardt, St. Louis, MO US

Patent applications by Lowell L. Wood, Jr., Bellevue, WA US

Patent applications by Muriel Y. Ishikawa, Livermore, CA US

Patent applications by Roderick A. Hyde, Redmond, WA US

Patent applications by Stephen L. Malaska, Redmond, WA US

Patent applications by Victoria Y.h. Wood, Livermore, CA US

Patent applications by Searete LLC, a limited liability corporation of the state of Delaware

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